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University of Illinois – Urbana-Champaign
1.
Srinivasan, Divya.
Single molecule pull-down of viral RNA-protein complexes.
Degree: MS, 0318, 2012, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/31924 
► Protein and RNA molecules interact with multiple protein partners to perform essential cellular processes such as post-transcriptional regulation of mRNA. Recently, a single molecule pull-down…
(more)
▼ Protein and RNA molecules interact with multiple protein partners to perform
essential cellular processes such as post-transcriptional regulation of mRNA. Recently,
a single molecule pull-down (SiMPull) assay was developed to isolate and study single
protein complexes directly from cell lysates. Unlike ensemble measurements, SiMPull is
a powerful tool that allows detection of diverse proteins present in a single complex and
quantitation of the number of interacting partners when the proteins are
stoichiometrically labeled. Using a similar principle, the objective of this study is to
extend the SiMPull assay to isolate and study single cellular RNA-protein complexes.
Utilizing a biological system of virally infected mammalian cells, the substrate targeted
in the study are viral RNA-protein complexes. Specifically, the highly replicative sendai
virus sub-genomic defective interfering (DI) RNA is targeted. The viral DI RNA
associates with multiple viral proteins during replication, and is therefore expected to
form heterogeneous RNA-protein complexes. Using the RNA sequence information of
DI RNA, we designed several short complementary DNA probes to capture single DI
RNA molecules for detection with single molecule fluorescence microscopy. We
demonstrate specific capture of viral DI RNA molecules using SiMPull, and could
quantitatively measure the presence of interacting viral proteins. Therefore, this study
provides evidence for the applicability of SiMPull to isolate and study single cellular
RNA-protein interactions.
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">
Ha,
Taekjip (advisor).
Subjects/Keywords: single molecule; viral RNA; total internal reflection fluorescence microscopy; Ribonucleic acid (RNA)
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APA (6th Edition):
Srinivasan, D. (2012). Single molecule pull-down of viral RNA-protein complexes. (Thesis). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/31924
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Srinivasan, Divya. “Single molecule pull-down of viral RNA-protein complexes.” 2012. Thesis, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/31924.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Srinivasan, Divya. “Single molecule pull-down of viral RNA-protein complexes.” 2012. Web. 01 Mar 2021.
Vancouver:
Srinivasan D. Single molecule pull-down of viral RNA-protein complexes. [Internet] [Thesis]. University of Illinois – Urbana-Champaign; 2012. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/31924.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Srinivasan D. Single molecule pull-down of viral RNA-protein complexes. [Thesis]. University of Illinois – Urbana-Champaign; 2012. Available from: http://hdl.handle.net/2142/31924
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
University of Illinois – Urbana-Champaign
2.
Rahil, Zainab.
Cellular decision making at the nanoscale.
Degree: MS, Bioengineering, 2016, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/98323
► The well-established dependence of cell traction forces on the compliance of supporting matrices has been attributed to levels of force exerted on components in focal…
(more)
▼ The well-established dependence of cell traction forces on the compliance of supporting matrices has been attributed to levels of force exerted on components in focal contacts. Here, use of novel, force-limited nanoscale tension gauges revealed that both force and substrate deformations govern cell decision-making during initial attachment to compliant substrates. We propose a mechanical model consistent with observed behavior. Upon formation of stable cell contacts, bond tension and tether rupture govern cell attachment, spreading, and focal adhesion maturation at force levels on individual receptors predicted by prior studies.
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Leckband%2C%20Deborah%22%29&pagesize-30">Leckband, Deborah (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (committee member).
Subjects/Keywords: Cell adhesion
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APA (6th Edition):
Rahil, Z. (2016). Cellular decision making at the nanoscale. (Thesis). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/98323
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Rahil, Zainab. “Cellular decision making at the nanoscale.” 2016. Thesis, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/98323.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Rahil, Zainab. “Cellular decision making at the nanoscale.” 2016. Web. 01 Mar 2021.
Vancouver:
Rahil Z. Cellular decision making at the nanoscale. [Internet] [Thesis]. University of Illinois – Urbana-Champaign; 2016. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/98323.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Rahil Z. Cellular decision making at the nanoscale. [Thesis]. University of Illinois – Urbana-Champaign; 2016. Available from: http://hdl.handle.net/2142/98323
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
University of Illinois – Urbana-Champaign
3.
Brenner, Michael.
Force manipulation and single molecule FRET of transcriptional regulatory factors.
Degree: PhD, 0335, 2013, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/42145
► Mechanical tension plays a large role in cell development ranging from morphology to gene expression. On the molecular level, the effects of tension can be…
(more)
▼ Mechanical tension plays a large role in cell development ranging from morphology to
gene expression. On the molecular level, the effects of tension can be seen in the dynamic
arrangement of membrane proteins as well as the recruitment and activation of intracellular
proteins leading to downstream signaling cascades regulating transcription. Forces applied to
biopolymers during in vitro force measurements offer greater understanding of the effects of
tension on molecules in live cells, and experimental techniques in test tubes and live cells can
often overlap. Indeed, when forces exerted on cellular components can be calibrated ex vivo with
force spectroscopy, a powerful tool is available for researchers in probing cellular
mechanotransduction on the molecular scale. Here we report the effect of peptide length on the
tension sensing properties of GPGGA peptide repeats using single-molecule fluorescence-force
spectroscopy. Additionally, we report on the mechanical properties of IκBα, a transcriptional
regulator, and the C-terminal domain of RNA polymerase II. Modification of proteins and
peptides for single-molecule studies was extended to incorporation of unnatural amino acids into
a DNA helicase. Chemical modification of RNA was performed to enable total-internal
reflection microscopy of single molecules of the guanine riboswitch aptamer domain, which is
involved in transcription termination. The combined FRET data support a model in which the
unfolded state of the aptamer domain has a highly dynamic P2 helix that switches rapidly
between two orientations relative to nondynamic P1 and P3. At <<1 mM Mg2+ (in the presence
of saturating guanine) or 1 mM Mg2+ (in the absence of guanine), the riboswitch starts to adopt
a folded conformation in which loop-loop interactions lock P2 and P3 into place. Another
transcription terminator, Rho helicase, was studied using single molecule techniques. Our
observations confirm the tethered-tracking model for RNA-directed Rho motion and suggest a
repetitive translocation mechanism involving reversible, step-wise threading of RNA through the
central Rho cavity in discrete steps, leading to loop formation at the exit side of the cavity. Our
data reveal that secondary structure and lower UC content of RNA impedes processive
translocation and results in more backwards motion of Rho helicase. We propose a global model
for Rho dynamics. Furthermore, these results provide general insights into the mechanisms of
RecA-family helicases and ring-shaped ATPases. Preliminary studies with the human
Argonaute2 nuclease will also be presented.
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">
Ha,
Taekjip (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Schulten%2C%20Klaus%20J.%22%29&pagesize-30">Schulten, Klaus J. (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Katzenellenbogen%2C%20John%20A.%22%29&pagesize-30">Katzenellenbogen, John A. (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Selvin%2C%20Paul%20R.%22%29&pagesize-30">Selvin, Paul R. (committee member).
Subjects/Keywords: transcription; single-molecule; optical tweezers; Förster Resonance Energy Transfer (FRET); biophysics
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APA (6th Edition):
Brenner, M. (2013). Force manipulation and single molecule FRET of transcriptional regulatory factors. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/42145
Chicago Manual of Style (16th Edition):
Brenner, Michael. “Force manipulation and single molecule FRET of transcriptional regulatory factors.” 2013. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/42145.
MLA Handbook (7th Edition):
Brenner, Michael. “Force manipulation and single molecule FRET of transcriptional regulatory factors.” 2013. Web. 01 Mar 2021.
Vancouver:
Brenner M. Force manipulation and single molecule FRET of transcriptional regulatory factors. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2013. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/42145.
Council of Science Editors:
Brenner M. Force manipulation and single molecule FRET of transcriptional regulatory factors. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2013. Available from: http://hdl.handle.net/2142/42145
University of Illinois – Urbana-Champaign
4.
Lee, Kyung Suk.
Fluorescence imaging of single molecule dynamics on long single stranded DNA.
Degree: PhD, 0240, 2013, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/42452
► Recent advances in single-molecule fluorescence imaging techniques have allowed the direct observation of protein dynamics on DNA, but the progress has been largely limited to…
(more)
▼ Recent advances in single-molecule fluorescence imaging techniques have allowed the direct observation of protein dynamics on DNA, but the progress has been largely limited to double-stranded DNA (dsDNA) or short single-stranded DNA (ssDNA). Here, a single molecule imaging approach for observing dynamics of proteins on long ssDNA of thousands of nucleotides in length is presented, with a detailed explanation on how to implement, operate and calibrate the hybrid instrument combining single-molecule fluorescence and force spectroscopy by which we could visualize the dynamics of proteins interacting with a long ssDNA similar in length to what’s generated during DNA metabolic processes. Also, various single-stranded DNA synthesis methods are included. The ultimate goal is to study many different proteins binding to the same long ssDNA and carrying out their function in coordination with each other. As a step toward the goal, here, the applicability of our methods to the dynamics of such ssDNA-interacting proteins is demonstrated by visualizing the dynamics of the following three proteins: (1) the DNA homology search on Escherichia coli (E. coli) RecA filaments formed on ssDNA, (2) the unidirectional motion of E. coli UvrD helicase and (3) the diffusion of E. coli single-stranded DNA binding protein (SSB). We observed that the homology search of dsDNA on RecA filament is comprised of pauses at certain sites followed by fast transitions between the sticky sites via 1D diffusion. As for UvrD, with the multidimensional data obtained with our platform, we could capture the entire sequence of binding, translocation, unwinding initiation of UvrD helicase with single molecule resolution. With SSB, we found that the diffusion coefficient is three orders of magnitude higher than what was determined from SSB diffusion on short ssDNA suggesting that on long ssDNA that mimics physiological setting, SSB can migrate via a long range intersegmental transfer. Force dependence of diffusion further supports the interpretation.
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">
Ha,
Taekjip (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Chemla%2C%20Yann%20R.%22%29&pagesize-30">Chemla, Yann R. (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Dahmen%2C%20Karin%20A.%22%29&pagesize-30">Dahmen, Karin A. (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Stack%2C%20John%20D.%22%29&pagesize-30">Stack, John D. (committee member).
Subjects/Keywords: Fluorescence; Optical Trap; Iamging; single stranded DNA (ssDNA)
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Lee, K. S. (2013). Fluorescence imaging of single molecule dynamics on long single stranded DNA. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/42452
Chicago Manual of Style (16th Edition):
Lee, Kyung Suk. “Fluorescence imaging of single molecule dynamics on long single stranded DNA.” 2013. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/42452.
MLA Handbook (7th Edition):
Lee, Kyung Suk. “Fluorescence imaging of single molecule dynamics on long single stranded DNA.” 2013. Web. 01 Mar 2021.
Vancouver:
Lee KS. Fluorescence imaging of single molecule dynamics on long single stranded DNA. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2013. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/42452.
Council of Science Editors:
Lee KS. Fluorescence imaging of single molecule dynamics on long single stranded DNA. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2013. Available from: http://hdl.handle.net/2142/42452
University of Illinois – Urbana-Champaign
5.
Vafabakhsh, Reza.
Single molecule fluorescence studies of biomolecular interactions.
Degree: PhD, 0240, 2013, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/44466
► Single molecule fluorescent techniques have become standard approaches to study protein-DNA interactions. However, these techniques have largely been confined by limitations in assays to studying…
(more)
▼ Single molecule fluorescent techniques have become standard approaches to study protein-DNA interactions. However, these techniques have largely been confined by limitations in assays to studying the interaction between simple DNA substrates and a single protein. During my PhD I developed several novel assays to study a long-standing controversial biophysics question (flexibility of short dsDNA), genome packaging in viruses (Influenza and T4) and dynamics of challenging protein complexes (membrane proteins).
The classical view of DNA posits that DNA must be stiff below the persistence length (<150 base pair) but recent studies addressing this have yielded contradictory results. We developed a fluorescence-based, protein-free, assay for studying the cyclization of single DNA molecules in real time. The looping rate for short DNA molecules has remarkably weak length dependence between 67 and 106 bps, deviating significantly from the worm-like chain model. We propose that many biologically significant protein-DNA interactions that involve looping and bending of DNA below 100 bp likely use this intrinsic bendability of DNA.
One of the critical aspects of a virus life cycle is packaging of the viral genome. Different viruses have devised intelligent mechanisms to perform this task. I studied packaging mechanism in Bacteriophage T4 and Influenza. Influenza A virus possesses a segmented genome of eight, single-stranded RNAs. However, the exact copy number of each viral RNA segment per individual virus particles has been controversial for the past 50 years. To address this question we combined single molecule TIRF microscopy and multi-color fluorescent in situ hybridization (FISH) to study the composition of viral RNAs at single-virus particle resolution. Our results showed that a high percentage of virus particles package a single copy of each segment of viral RNAs. Our findings support a model that the packaging of influenza genome is a selective and robust process.
Finally we developed a single molecule fluorescence assay to study initiation and re-initiation of dsDNA packaging in the T4 bacteriophage. Using this assay we quantified the details of T4 “packasome” assembly. Also, we showed that the T4 packaging machine can package multiple DNA into the same head in burst-like fashion.
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">
Ha,
Taekjip (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Goldenfeld%2C%20Nigel%20D.%22%29&pagesize-30">Goldenfeld, Nigel D. (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Stack%2C%20John%20D.%22%29&pagesize-30">Stack, John D. (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Chemla%2C%20Yann%20R.%22%29&pagesize-30">Chemla, Yann R. (committee member).
Subjects/Keywords: Single molecule; Fluorescence Resonance Energy Transfer (FRET); Deoxyribonucleic Acid (DNA); Influenza; bacteriophage T4 packaging
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APA ·
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MLA ·
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Export
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APA (6th Edition):
Vafabakhsh, R. (2013). Single molecule fluorescence studies of biomolecular interactions. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/44466
Chicago Manual of Style (16th Edition):
Vafabakhsh, Reza. “Single molecule fluorescence studies of biomolecular interactions.” 2013. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/44466.
MLA Handbook (7th Edition):
Vafabakhsh, Reza. “Single molecule fluorescence studies of biomolecular interactions.” 2013. Web. 01 Mar 2021.
Vancouver:
Vafabakhsh R. Single molecule fluorescence studies of biomolecular interactions. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2013. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/44466.
Council of Science Editors:
Vafabakhsh R. Single molecule fluorescence studies of biomolecular interactions. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2013. Available from: http://hdl.handle.net/2142/44466
University of Illinois – Urbana-Champaign
6.
Syed, Salman.
Single molecule fluorescence studies of replicative helicases.
Degree: PhD, 0319, 2014, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/46948
► DNA helicases are motor enzymes that convert the chemical energy of nucleotide triphosphate hydrolysis into mechanical energy for translocation on single stranded (ss) DNA and…
(more)
▼ DNA helicases are motor enzymes that convert the chemical energy of nucleotide triphosphate hydrolysis into mechanical energy for translocation on single stranded (ss) DNA and unwinding of double stranded (ds) DNA. All of the 12 known replicative helicases are hexameric helicases that are ring-shaped in structure and are homohexamers with the exception of the eukaryotic minichromosomal maintenance (MCM) helicase. Their activities are essential for a variety of DNA metabolic transactions including replication, recombination and repair. Bacteriophage T7 gp4 serves as one of the model proteins for replicative helicases. Moreover, the replication machinery of Bacteriophage T7 provides a simple model to study the dynamics of DNA replication. To address how the leading and lagging strand synthesis are coordinated during DNA replication, we employed single molecule Förster Resonance Energy Transfer (smFRET) methods on the Bacteriophage T7 replisome (helicase/primase and polymerase). Our results suggest a model whereby the primase loop allows the coordination of leading and lagging strand DNA synthesis by enabling the newly synthesized RNA primers to remain coupled to the leading strand replication complex and therefore minimizing the delay in lagging strand synthesis. To further investigate the unwinding mechanism of T7 helicase, we used smFRET methods to resolve steps during DNA unwinding by T7 helicase. Our results show direct evidence of larger than 1 bp step size of unwinding by a hexameric helicase. Our studies provide a detailed mechanism of T7 helicase unwinding of double stranded (ds) DNA.
Simian Virus (SV) 40 Large T-Antigen (L-tag) serves as a model replicative helicase for studying eukaryotic DNA replication. It belongs to the AAA+ family of helicases that bind to the origin of replication sites on the DNA and form higher order structures to unwind the DNA bidirectionally. We used smFRET methods to probe the unwinding activity of L-tag. An important aspect in understanding the translocation and unwinding mechanisms of ring helicases is how the subunits of these enzymes are coordinated during ATP hydrolysis cycles as they move on ss/dsDNA. E1, another helicase belonging to the AAA+ family of helicases, was shown to utilize a sequential ATP hydrolysis mechanism during ssDNA translocation where individual subunits hydrolyze ATP one at a time. For L-tag, it has been proposed that it utilizes a concerted ATP hydrolysis mechanism where all the subunits hydrolyze ATP simultaneously. To address this question, we performed experiments using L-tag proteins that are deficient in their ATPase and DNA binding activities at different ratios with the wild type L-tag. Our results suggest that individual subunits of the hexamer are coordinated during ATP hydrolysis but do not differentiate between a concerted vs. sequential ATP hydrolysis mechanism.
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">
Ha,
Taekjip (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Nair%2C%20Satish%20K.%22%29&pagesize-30">Nair, Satish K. (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Chemla%2C%20Yann%20R.%22%29&pagesize-30">Chemla, Yann R. (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Myong%2C%20Su-A%22%29&pagesize-30">Myong, Su-A (committee member).
Subjects/Keywords: Single Molecule Fluorescence T7 Helicase
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APA ·
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MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Syed, S. (2014). Single molecule fluorescence studies of replicative helicases. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/46948
Chicago Manual of Style (16th Edition):
Syed, Salman. “Single molecule fluorescence studies of replicative helicases.” 2014. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/46948.
MLA Handbook (7th Edition):
Syed, Salman. “Single molecule fluorescence studies of replicative helicases.” 2014. Web. 01 Mar 2021.
Vancouver:
Syed S. Single molecule fluorescence studies of replicative helicases. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2014. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/46948.
Council of Science Editors:
Syed S. Single molecule fluorescence studies of replicative helicases. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2014. Available from: http://hdl.handle.net/2142/46948
University of Illinois – Urbana-Champaign
7.
Diao, Jiajie.
Single-molecule fluorescence resonance energy transfer study of SNARE-mediated membrane fusion.
Degree: PhD, 0240, 2011, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/18580
► This is a comprehensive study of protein-mediated membrane fusion through single-molecule fluorescence resonance energy transfer (smFRET). Membrane fusion is one of the important cellular processes…
(more)
▼ This is a comprehensive study of protein-mediated membrane fusion through single-molecule fluorescence resonance energy transfer (smFRET). Membrane fusion is one of the important cellular processes by which two initially distinct lipid bilayers merge their hydrophobic cores, resulting in one interconnected structure. For example, exocytosis, fertilization of an egg by a sperm and communication between neurons are a few among many processes that rely on some form of fusion. Proteins called soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) play a central role in fusion processes which is also regulated by many accessory proteins, such as synaptotagmin, complexin and Munc18.
By a new lipid mixing method at the single-vesicle level, we are able to accurately detect different stages of SNARE-mediated membrane fusion including docking, hemi and full fusion via FRET value of single donor/acceptor vesicle pair. Through this single-vesicle lipid mixing assay, we discovered the vesicle aggregation induced by C2AB/Ca2+, the dual function of complexin, and the fusion promotion role of Munc18/SNARE-core binding mode.
While this new method provides the information regarding the extent of the ensemble lipid mixing, the fusion pore opening between two vesicular cavities and the interaction between proteins cannot be detected. In order to overcome these limitations, we then developed a single-vesicle content mixing method to reveal the key factor of pore expansion by detecting the FRET change of dual-labeled DNA probes encapsulated in vesicles. Through our single-vesicle content mixing assay, we found the fusion pore expansion role of yeast SNAREs as well as neuronal SNAREs plus synaptotagmin 1.
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">
Ha,
Taekjip (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Chemla%2C%20Yann%20R.%22%29&pagesize-30">Chemla, Yann R. (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Rogers%2C%20John%20A.%22%29&pagesize-30">Rogers, John A. (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Aksimentiev%2C%20Aleksei%22%29&pagesize-30">Aksimentiev, Aleksei (committee member).
Subjects/Keywords: Single molecule; Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE); Membrane fusion
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APA ·
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APA (6th Edition):
Diao, J. (2011). Single-molecule fluorescence resonance energy transfer study of SNARE-mediated membrane fusion. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/18580
Chicago Manual of Style (16th Edition):
Diao, Jiajie. “Single-molecule fluorescence resonance energy transfer study of SNARE-mediated membrane fusion.” 2011. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/18580.
MLA Handbook (7th Edition):
Diao, Jiajie. “Single-molecule fluorescence resonance energy transfer study of SNARE-mediated membrane fusion.” 2011. Web. 01 Mar 2021.
Vancouver:
Diao J. Single-molecule fluorescence resonance energy transfer study of SNARE-mediated membrane fusion. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2011. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/18580.
Council of Science Editors:
Diao J. Single-molecule fluorescence resonance energy transfer study of SNARE-mediated membrane fusion. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2011. Available from: http://hdl.handle.net/2142/18580
University of Illinois – Urbana-Champaign
8.
Ngo, Thuy.
Regulation of nucleosome dynamics.
Degree: PhD, 0319, 2015, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/73070
► In eukaryotic cells, the genome is packed into fundamental units called nucleosomes, where 147 base pairs of DNA are wrapped around a protein core. Stable…
(more)
▼ In eukaryotic cells, the genome is packed into fundamental units called nucleosomes, where 147 base pairs of DNA are wrapped around a protein core. Stable packing of DNA in nucleosomes imposes a barrier for accessibility of genetic code on DNA for replication, transcription, and repair. The dynamics of nucleosomal DNA provide a mean for gene regulation by genomic sequence and epigenetic modifications. Understanding the physical basis of how sequence and epigenetic modifications of DNA affect nucleosome dynamics and nucleosomal DNA exposure will help elucidate how genomic and epigenetic modifications regulate cellular functions, cell differentiation and cancer development.
This motivated us to investigate local conformational dynamics of the nucleosome under tension or in the relaxed state and its modulation by DNA sequence and modifications. We achieved the goals by utilizing single-molecule force fluorescence spectroscopy, which allows monitoring dynamics of nucleosome at a define locality, and single-molecule DNA cyclization measurements, which enables determining of correlation of nucleosome dynamics with DNA flexibility. Chapter 2 shows details of sample preparation and methods used in studies of this dissertation.
We made three profound discoveries: (1) the nucleosome unwraps directionally under force; (2) DNA flexibility is the basic physical property that controls nucleosome stability and nucleosomal DNA accessibility; and (3) two nucleosomal DNA ends are orchestrated such that the opening of one end helps stabilize the other end, providing a mechanism to amplify even a small difference in flexibility to a large mechanical asymmetry. These results are presented in Chapter 3.
In Chapter 4 and 5, we present results to further demonstrate the correlation between DNA flexibility and unwrapping force by varying DNA modifications such as DNA mismatches, 5-methylcytosine and 5-formylcytosine. DNA methylation (5-methylcytosine) decreases DNA flexibility and reduces nucleosome mechanical stability while DNA mismatches and 5-formylcytosine have opposite effects. Our results suggest a completely new mechanism through which DNA sequence and epigenetic marks on DNA may be utilized to regulate gene expression by controlling nucleosome accessibility for replication, transcription, repair and remodeling.
Finally, we identified slow spontaneous local gaping of nucleosomes under physiological conditions. Gaping modes switch along the direction normal to the DNA plane at minutes (1-10 minutes) time scale. The existence of nucleosome in different gaping modes may underlie the heterogeneous enzymatic reactions on chromatin substrates and the formation of multiple compression forms of chromatin fibers. These results are detailed in Chapter 6.
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">
Ha,
Taekjip (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Belmont%2C%20Andrew%20S.%22%29&pagesize-30">Belmont, Andrew S. (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Chemla%2C%20Yann%22%29&pagesize-30">Chemla, Yann (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Aksimentiev%2C%20Aleksei%22%29&pagesize-30">Aksimentiev, Aleksei (committee member).
Subjects/Keywords: nucleosomes; unwrapping; nucleosome dynamics; asymmetry; DNA sequence; DNA flexibility; DNA modifications; optical tweezers; fluorescence resonance energy transfer (FRET); single-molecule; DNA cyclization; gaping; DNA methylation (5-mC); 5-Hydroxymethylcytosine (5-hmC); 5-formylcytosine (5-fC); 5-carboxylcytosine (5-caC); mismatch
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APA (6th Edition):
Ngo, T. (2015). Regulation of nucleosome dynamics. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/73070
Chicago Manual of Style (16th Edition):
Ngo, Thuy. “Regulation of nucleosome dynamics.” 2015. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/73070.
MLA Handbook (7th Edition):
Ngo, Thuy. “Regulation of nucleosome dynamics.” 2015. Web. 01 Mar 2021.
Vancouver:
Ngo T. Regulation of nucleosome dynamics. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2015. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/73070.
Council of Science Editors:
Ngo T. Regulation of nucleosome dynamics. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2015. Available from: http://hdl.handle.net/2142/73070
University of Illinois – Urbana-Champaign
9.
Zhou, Ruobo.
Fluorescence-force spectroscopy at the single molecule level.
Degree: PhD, 0240, 2012, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/31921
► During the past decade, various powerful single-molecule techniques have evolved and helped to address important questions in life sciences. As the single molecule techniques become…
(more)
▼ During the past decade, various powerful single-molecule techniques have
evolved and helped to address important questions in life sciences. As the single
molecule techniques become mature, there is increasingly pressing need to maximize the
information content of the analysis in order to be able to study more complex systems
that better approximate in-vivo conditions. Here, we develop a fluorescence-force
spectroscopy method to combine single-molecule fluorescence spectroscopy with optical
tweezers. Optical tweezers are used to manipulate and observe mechanical properties on
the nanometer scale and piconewton force range. However, once the force range is in the
low piconewton range or less, the spatial resolution of optical tweezers decreases
significantly. In combination with fluorescence spectroscopy, like single molecule
Förster (or fluorescence) resonance energy transfer (FRET) whose detectable distance
range is approximately 3-10 nm, we are able to observe nanometer fluctuations and
internal conformational changes in a low-force regime. The possibility to place
fluorescent labels at nearly any desired position and a sophisticated design of the
experiment increases the amount of information that can be extracted in contrast to pure
mechanical or fluorescence experiments. We demonstrate the applications of this method
to various biological systems including: 1) to measure the effect of very low forces on the
nanometer scale conformational transitions of the DNA four-way (Holliday) junction; 2)
to dissect protein diffusion and dissociation mechanisms on single stranded DNA, 3) to
calibrate FRET-based in-vivo force sensors and 4) to study mechanical unfolding of
single proteins. The results could not have been obtained with fluorescence or force
measurement alone, and clearly demonstrates the power and generality of our approach.
Finally, we show that self-quenching of two identical fluorophores can be used to detect
small conformational dynamics corresponding to sub-nanometer distance changes of
single molecules in a FRET-insensitive short range (< 3 nm), extending the detectable
distance range of our fluorescence-force spectroscopy method.
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">
Ha,
Taekjip (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Aksimentiev%2C%20Aleksei%22%29&pagesize-30">Aksimentiev, Aleksei (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Selvin%2C%20Paul%20R.%22%29&pagesize-30">Selvin, Paul R. (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Stack%2C%20John%20D.%22%29&pagesize-30">Stack, John D. (committee member).
Subjects/Keywords: single molecule detection; fluorescence microscopy; optical tweezers
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Zhou, R. (2012). Fluorescence-force spectroscopy at the single molecule level. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/31921
Chicago Manual of Style (16th Edition):
Zhou, Ruobo. “Fluorescence-force spectroscopy at the single molecule level.” 2012. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/31921.
MLA Handbook (7th Edition):
Zhou, Ruobo. “Fluorescence-force spectroscopy at the single molecule level.” 2012. Web. 01 Mar 2021.
Vancouver:
Zhou R. Fluorescence-force spectroscopy at the single molecule level. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2012. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/31921.
Council of Science Editors:
Zhou R. Fluorescence-force spectroscopy at the single molecule level. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2012. Available from: http://hdl.handle.net/2142/31921
University of Illinois – Urbana-Champaign
10.
Madabhusi Ragunathan, Kaushik.
Single molecule fret study on the mechanism of RecA mediated strand exchange.
Degree: PhD, 0319, 2012, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/32044
► RecA plays a critical role during double strand break repair via homologous recombination. During the strand exchange reaction, RecA forms a helical filament on single…
(more)
▼ RecA plays a critical role during double strand break repair via homologous recombination. During the strand exchange reaction, RecA forms a helical filament on single stranded (ss) DNA that searches for homology and exchanges complementary base pairs with a homologous double strand (ds) DNA to form a new heteroduplex. The study of strand exchange in ensemble assays is limited by the diffusion limited homology search process which masks the subsequent strand exchange reaction. We developed a single molecule fluorescence assay with a few basepair and milliseconds resolution which can separate initial docking from the subsequent propagation of joint molecule formation. Our data suggests that propagation occurs in 3 bp increments with destabilization of the incoming dsDNA and concomitant pairing with the reference ssDNA. Our model for strand exchange links structural models of RecA to its catalytic function.
Next, we investigated the mechanism of RecA mediated homology search. Using tools with high spatiotemporal resolution to observe the encounter complex between the RecA filament and dsDNA, we present evidence in support of the “sliding model” wherein a RecA filament diffuses on a dsDNA track. Our results suggest that the sliding of the dsDNA relative to the RecA filament can explain the rapid changes in FRET which we have observed upon the docking of non-homologous dsDNA to the RecA filament. We further show that homology can be identified during such sliding. Sliding is thermally driven and occurs in the absence of ATP hydrolysis. Furthermore, homology recognition and basepairing can involve as few as 6 bp of complementarity. Our observation presents an example of how a multi-protein complex bound to DNA can serve as a vehicle enabling homology search processes via 1-D sliding.
Finally, we demonstrate how an extension of the two color FRET assay to measure four colors simultaneously allows us to measure the correlation of reaction completion between the two ends of a single synaptic complex. We expect that this method will enable a multi dimensional analysis of independent reaction coordinates with broad applications in measuring the correlated dynamics of more complex biological systems
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">
Ha,
Taekjip (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Nair%2C%20Satish%20K.%22%29&pagesize-30">Nair, Satish K. (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Spies%2C%20Maria%22%29&pagesize-30">Spies, Maria (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Chemla%2C%20Yann%20R.%22%29&pagesize-30">Chemla, Yann R. (committee member).
Subjects/Keywords: DNA repair; recombination; single molecule; Fluorescence resonance energy transfer (FRET); fluorescence; Deoxyribonucleic acid (DNA)
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Madabhusi Ragunathan, K. (2012). Single molecule fret study on the mechanism of RecA mediated strand exchange. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/32044
Chicago Manual of Style (16th Edition):
Madabhusi Ragunathan, Kaushik. “Single molecule fret study on the mechanism of RecA mediated strand exchange.” 2012. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/32044.
MLA Handbook (7th Edition):
Madabhusi Ragunathan, Kaushik. “Single molecule fret study on the mechanism of RecA mediated strand exchange.” 2012. Web. 01 Mar 2021.
Vancouver:
Madabhusi Ragunathan K. Single molecule fret study on the mechanism of RecA mediated strand exchange. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2012. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/32044.
Council of Science Editors:
Madabhusi Ragunathan K. Single molecule fret study on the mechanism of RecA mediated strand exchange. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2012. Available from: http://hdl.handle.net/2142/32044
University of Illinois – Urbana-Champaign
11.
Arslan, Sinan.
Exploration of helicase mechanisms at single molecule level: answering decades old questions and bioengineering super proteins.
Degree: PhD, Physics, 2014, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/94165
► In the last 15 years, single molecule techniques have allowed us to observe the transient, heterogeneous, and multi‐step behavior of biomolecules often averaged out and…
(more)
▼ In the last 15 years, single molecule techniques have allowed us to observe the transient, heterogeneous, and multi‐step behavior of biomolecules often averaged out and lost in ensemble assays. Helicases form a ubiquitous class of enzymes that function in many aspects of nucleic acid metabolism which is central to life. We used single molecule Förster resonance energy transfer (FRET) and optical tweezers force spectroscopy to study translocation and unwinding mechanism of Escherichia coli Rep, a model helicase with 3’5’ motor translocation activity on single‐stranded DNA (ssDNA) fueled by ATP hydrolysis. When a translocating Rep on a 3’‐overhang DNA reaches the DNA duplex junction, instead of dissociating, it snaps back to the 3’ end, restarting another shuttling cycle. We investigated the mechanism of repetitive shuttling and discovered the same behavior on various DNA substrates. We concluded that the repetitions are induced by the lack of ssDNA track ahead of the Rep. Using the repetitive shuttling assay, we explored the translocation mechanism of Rep in detail. First, we perturbed the ssDNA binding network via biochemical mutations that led to determination of the key residues that control shuttling speed, ATPase activity, and directionality. Second, we tested the effects of the DNA lesions on Rep translocation, observing that the irregularities encompassing 1‐3 nucleotides in the backbone and nucleobases only caused transient stalls. To probe the unwinding mechanism, we developed a conformational control assay which turned wild type Rep monomers with no detectable DNA unwinding activity into super‐helicases (Rep‐X) via internal covalent crosslinking. Rep‐X can unwind thousands of base pairs processively even against large forces. We also showed that partner proteins of a similar enzyme turn on the unwinding activity by stabilizing the active form. Lastly, the orientation dependence of FRET is experimentally shown between nucleic acid conjugated cyanine fluorophores that constitutes the first demonstration of this effect in a biophysical system since its formulation by Theodor Förster in 1948.
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">
Ha,
Taekjip (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Selvin%2C%20Paul%20R%22%29&pagesize-30">Selvin, Paul R (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Dahmen%2C%20Karin%20A%22%29&pagesize-30">Dahmen, Karin A (committee member),
Champaign%22%20%2Bcontributor%3A%28%22DeMarco%2C%20Brian%20L%22%29&pagesize-30">DeMarco, Brian L (committee member).
Subjects/Keywords: orientation dependence; protein function; conformational control; active conformation; protein engineering; helicase unwinding; DNA unwinding; repetitive translocation; repetitive shuttling; PcrA; Rep; super family 1 helicases; helicase; single molecule FRET; FRET; single molecule
Record Details
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Arslan, S. (2014). Exploration of helicase mechanisms at single molecule level: answering decades old questions and bioengineering super proteins. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/94165
Chicago Manual of Style (16th Edition):
Arslan, Sinan. “Exploration of helicase mechanisms at single molecule level: answering decades old questions and bioengineering super proteins.” 2014. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/94165.
MLA Handbook (7th Edition):
Arslan, Sinan. “Exploration of helicase mechanisms at single molecule level: answering decades old questions and bioengineering super proteins.” 2014. Web. 01 Mar 2021.
Vancouver:
Arslan S. Exploration of helicase mechanisms at single molecule level: answering decades old questions and bioengineering super proteins. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2014. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/94165.
Council of Science Editors:
Arslan S. Exploration of helicase mechanisms at single molecule level: answering decades old questions and bioengineering super proteins. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2014. Available from: http://hdl.handle.net/2142/94165
University of Illinois – Urbana-Champaign
12.
Arslan, Sinan.
Exploration of helicase mechanisms at single molecule level: answering decades old questions and bioengineering super proteins.
Degree: PhD, Physics, 2014, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/98320
► In the last 15 years, single molecule techniques have allowed us to observe the transient, heterogeneous, and multi‐step behavior of biomolecules often averaged out and…
(more)
▼ In the last 15 years, single molecule techniques have allowed us to observe the transient, heterogeneous, and multi‐step behavior of biomolecules often averaged out and lost in ensemble assays. Helicases form a ubiquitous class of enzymes that function in many aspects of nucleic acid metabolism which is central to life. We used single molecule Förster resonance energy transfer (FRET) and optical tweezers force spectroscopy to study translocation and unwinding mechanism of Escherichia coli Rep, a model helicase with 3’5’ motor translocation activity on single‐stranded DNA (ssDNA) fueled by ATP hydrolysis. When a translocating Rep on a 3’‐overhang DNA reaches the DNA duplex junction, instead of dissociating, it snaps back to the 3’ end, restarting another shuttling cycle. We investigated the mechanism of repetitive shuttling and discovered the same behavior on various DNA substrates. We concluded that the repetitions are induced by the lack of ssDNA track ahead of the Rep. Using the repetitive shuttling assay, we explored the translocation mechanism of Rep in detail. First, we perturbed the ssDNA binding network via biochemical mutations that led to determination of the key residues that control shuttling speed, ATPase activity, and directionality. Second, we tested the effects of the DNA lesions on Rep translocation, observing that the irregularities encompassing 1‐3 nucleotides in the backbone and nucleobases only caused transient stalls. To probe the unwinding mechanism, we developed a conformational control assay which turned wild type Rep monomers with no detectable DNA unwinding activity into super‐helicases (Rep‐X) via internal covalent crosslinking. Rep‐X can unwind thousands of base pairs processively even against large forces. We also showed that partner proteins of a similar enzyme turn on the unwinding activity by stabilizing the active form. Lastly, the orientation dependence of FRET is experimentally shown between nucleic acid conjugated cyanine fluorophores that constitutes the first demonstration of this effect in a biophysical system since its formulation by Theodor Förster in 1948.
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">
Ha,
Taekjip (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Selvin%2C%20Paul%20R%22%29&pagesize-30">Selvin, Paul R (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Dahmen%2C%20Karin%20A%22%29&pagesize-30">Dahmen, Karin A (committee member),
Champaign%22%20%2Bcontributor%3A%28%22DeMarco%2C%20Brian%20L%22%29&pagesize-30">DeMarco, Brian L (committee member).
Subjects/Keywords: orientation dependence; protein function; conformational control; active conformation; protein engineering; helicase unwinding; DNA unwinding; repetitive translocation; repetitive shuttling; PcrA; Rep; super family 1 helicases; helicase; single molecule FRET; FRET; single molecule
Record Details
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Record Details
Similar Records
Cite
« Share
❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Arslan, S. (2014). Exploration of helicase mechanisms at single molecule level: answering decades old questions and bioengineering super proteins. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/98320
Chicago Manual of Style (16th Edition):
Arslan, Sinan. “Exploration of helicase mechanisms at single molecule level: answering decades old questions and bioengineering super proteins.” 2014. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/98320.
MLA Handbook (7th Edition):
Arslan, Sinan. “Exploration of helicase mechanisms at single molecule level: answering decades old questions and bioengineering super proteins.” 2014. Web. 01 Mar 2021.
Vancouver:
Arslan S. Exploration of helicase mechanisms at single molecule level: answering decades old questions and bioengineering super proteins. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2014. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/98320.
Council of Science Editors:
Arslan S. Exploration of helicase mechanisms at single molecule level: answering decades old questions and bioengineering super proteins. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2014. Available from: http://hdl.handle.net/2142/98320
University of Illinois – Urbana-Champaign
13.
Stekas, Barbara.
The effect of single-stranded DNA binding protein RPA2 on XPD helicase processivity.
Degree: PhD, Physics, 2018, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/101311
► Understanding how proteins work together to perform vital cellular functions, such as replicating and repairing DNA, not only extends our understanding of fundamental biology, but…
(more)
▼ Understanding how proteins work together to perform vital cellular functions, such as replicating and repairing DNA, not only extends our understanding of fundamental biology, but can also lead to important medical interventions. As a necessary first step to understanding larger systems, we focus on a two-protein system involved in DNA repair. Xeroderma pigmentosum group D (XPD) is a helicase protein that plays an important role in nucleotide excision repair (NER). Its function is to unwind double-stranded DNA, allowing access to the bases that connect the strands and code genetic information. Previous work has shown that XPD activity is enhanced by the single-stranded DNA binding protein replication protein A (RPA2). However, the mechanism by which unwinding enhancement occurs is unknown. In single-molecule optical trapping experiments, we monitor – with single base-pair precision – the unwinding of a DNA hairpin by XPD in the presence of RPA. We observe the effect of RPA2 on XPD unwinding in real time and distinguish between proposed models of protein cooperation by analyzing changes in unwinding behavior with added RPA. Our data disfavor mechanisms by which RPA2 melts the duplex ahead of XPD as well as RPA2 sequestering ssDNA behind the helicase. We present our own 2-state kinetic model of XPD unwinding that we believe explains our data best. We propose that XPD has two inherent states of unwinding, high and low processivity, and that RPA2 aids unwinding by increasing the likelihood of XPD being in its more processive state.
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Chemla%2C%20Yann%20R%22%29&pagesize-30">Chemla, Yann R (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Aksimentiev%2C%20Aleksei%22%29&pagesize-30">Aksimentiev, Aleksei (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (committee member),
Champaign%22%20%2Bcontributor%3A%28%22DeMarco%2C%20Brian%22%29&pagesize-30">DeMarco, Brian (committee member).
Subjects/Keywords: helicase; XPD; RPA; processivity; optical trapping; optical tweezers; nucleotide excision repair; NER
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Record Details
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Stekas, B. (2018). The effect of single-stranded DNA binding protein RPA2 on XPD helicase processivity. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/101311
Chicago Manual of Style (16th Edition):
Stekas, Barbara. “The effect of single-stranded DNA binding protein RPA2 on XPD helicase processivity.” 2018. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/101311.
MLA Handbook (7th Edition):
Stekas, Barbara. “The effect of single-stranded DNA binding protein RPA2 on XPD helicase processivity.” 2018. Web. 01 Mar 2021.
Vancouver:
Stekas B. The effect of single-stranded DNA binding protein RPA2 on XPD helicase processivity. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2018. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/101311.
Council of Science Editors:
Stekas B. The effect of single-stranded DNA binding protein RPA2 on XPD helicase processivity. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2018. Available from: http://hdl.handle.net/2142/101311
University of Illinois – Urbana-Champaign
14.
Jain, Ankur.
Probing cellular protein complexes using single-molecule pull-down.
Degree: PhD, 0319, 2014, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/46887
► Cellular processes result from dynamic interactions between biomolecules. The gold standard method for investigating interaction between biomolecules is the pull-down assay. We have extended the…
(more)
▼ Cellular processes result from dynamic interactions between biomolecules. The gold standard method for investigating interaction between biomolecules is the pull-down assay. We have extended the conventional pull-down assay to its ultimate limit: the analysis of single biomolecular complexes. We achieve this goal by performing the pull-downs directly on to the surface of microscope slides and visualizing the captured biomolecules at single-molecule resolution using total internal reflection fluorescence (TIRF) microscopy. We name this technology single-molecule pull-down or SiMPull.
In one reification biotinylated antibody against the protein of interest is immobilized on a flow chamber. The flow chambers are passivated using a polymer coating such that the cellular components do not bind, and the immobilized antibody specifically captures the target protein. Cell lysates are diluted to obtain isolated molecules suitable for single-molecule imaging. The target molecules are fluorescently labeled either using a genetically encoded fluorescent protein tag or using antibodies, and imaged using a single-molecule TIRF microscope.
Using SiMPull we are able to discriminate between multiple association states of a protein as well as determine the stoichiometry of interaction. This technology is widely applicable to an array of biological contexts, and is suitable to analysis of endogenous protein complexes from animal tissue. In particular, we have used SiMPull to investigate the architecture and assembly of mechanistic target of rapamycin complexes.
The complexes captured from cell extracts on to our imaging chambers retain their functional activities: thus SiMPull can be used as a preparatory tool for single-molecule biochemical analysis on proteins that cannot be readily purified or reconstituted. Finally, we have extended this approach to the analysis of lipid-protein interactions.
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">
Ha,
Taekjip (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Chen%2C%20Jie%22%29&pagesize-30">Chen, Jie (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Gruebele%2C%20Martin%22%29&pagesize-30">Gruebele, Martin (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Myong%2C%20Su-A%22%29&pagesize-30">Myong, Su-A (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Prasanth%2C%20Supriya%20G.%22%29&pagesize-30">Prasanth, Supriya G. (committee member).
Subjects/Keywords: single-molecule; fluorescence microscopy; protein interactions
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APA (6th Edition):
Jain, A. (2014). Probing cellular protein complexes using single-molecule pull-down. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/46887
Chicago Manual of Style (16th Edition):
Jain, Ankur. “Probing cellular protein complexes using single-molecule pull-down.” 2014. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/46887.
MLA Handbook (7th Edition):
Jain, Ankur. “Probing cellular protein complexes using single-molecule pull-down.” 2014. Web. 01 Mar 2021.
Vancouver:
Jain A. Probing cellular protein complexes using single-molecule pull-down. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2014. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/46887.
Council of Science Editors:
Jain A. Probing cellular protein complexes using single-molecule pull-down. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2014. Available from: http://hdl.handle.net/2142/46887
University of Illinois – Urbana-Champaign
15.
Aref, Thomas.
Probing quantum phase slips in superconducting nanowires modified using high bias voltage pulses.
Degree: PhD, 0240, 2011, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/18460
► Quantum phase slips (QPS) or the macroscopic quantum tunneling (MQT) of a nanowire???s order parameter through an activation energy barrier have remained the subject of…
(more)
▼ Quantum phase slips (QPS) or the macroscopic quantum tunneling (MQT) of a nanowire???s order parameter through an activation energy barrier have remained the subject of intense debate for many years. They are expected to occur at low enough temperature where thermally activated phase slips (TAPS) have been frozen out in analogy with Josephson junctions where the macroscopic tunneling of phase at low temperatures has been conclusively experimentally demonstrated. We address this question by following a similar experimental strategy to that employed for establishing MQT in JJs. By measuring switching current distributions, we can probe the phase slip rate and determine if it corresponds to thermal activation or quantum tunneling. Having established that the behavior we see is consistent with being in the quantum regime, we can alter properties of the nanowires and see if the response is consistent with the expectations of the quantum model. To do this we employ an in-situ modification technique using high bias voltage pulses. Using these pulses, we can change resistance, critical temperature, critical current and morphology of the nanowire. We can also change the shunting capacitance of the nanowire by altering the photolithography step used to create the nanowires electrodes. The resulting response of the nanowires agrees well with being in the quantum tunneling dominated regime. An interesting side benefit of the pulsing technique is that we can exactly set the nanowires switching current to a desired value. This may be instrumental in the development of superconducting nanowire qubits.
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Bezryadin%2C%20Alexey%22%29&pagesize-30">Bezryadin, Alexey (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Eckstein%2C%20James%20N.%22%29&pagesize-30">Eckstein, James N. (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Bezryadin%2C%20Alexey%22%29&pagesize-30">Bezryadin, Alexey (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Oono%2C%20Yoshitsugu%22%29&pagesize-30">Oono, Yoshitsugu (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (committee member).
Subjects/Keywords: quantum phase slip; superconducting nanowires; electromigration; pulsing; high bias voltage; QPS; TAPS; phase slip
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APA ·
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MLA ·
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Export
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APA (6th Edition):
Aref, T. (2011). Probing quantum phase slips in superconducting nanowires modified using high bias voltage pulses. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/18460
Chicago Manual of Style (16th Edition):
Aref, Thomas. “Probing quantum phase slips in superconducting nanowires modified using high bias voltage pulses.” 2011. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/18460.
MLA Handbook (7th Edition):
Aref, Thomas. “Probing quantum phase slips in superconducting nanowires modified using high bias voltage pulses.” 2011. Web. 01 Mar 2021.
Vancouver:
Aref T. Probing quantum phase slips in superconducting nanowires modified using high bias voltage pulses. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2011. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/18460.
Council of Science Editors:
Aref T. Probing quantum phase slips in superconducting nanowires modified using high bias voltage pulses. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2011. Available from: http://hdl.handle.net/2142/18460
University of Illinois – Urbana-Champaign
16.
Maffeo, Christopher.
Quantitative all-atom and coarse-grained molecular dynamics simulation studies of DNA.
Degree: PhD, 0240, 2015, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/72871
► The remarkable molecule that encodes genetic information for all life on earth—DNA—is a polymer with unusual physical properties. The mechanical and electrostatic properties of DNA…
(more)
▼ The remarkable molecule that encodes genetic information for all life on earth—DNA—is a polymer with unusual physical properties. The mechanical and electrostatic properties of DNA are utilized extensively by cells in the replication, regular maintenance, and expression of their genetic material. This can be illustrated by considering the journey of a typical gene regulating protein, the lac repressor, which recognizes a particular gene and prevents its expression. First, the large electrostatic charge density of DNA provides an energetic track that guides the repressor’s search for its target binding site. Next, as the protein moves along the DNA, it attempts to deform the DNA. The repressor is only able to form an active complex with DNA that has the right sequence-dependent flexibility. Finally, the repressor is believed to form a very small DNA loop that prevents the gene from being expressed. The stability of the loop can be expected to depend sensitively on the global flexibility of DNA. Thus, the key to understanding the some of the most important cellular processes lies in understanding the physical properties of DNA. Single-molecule experiments allow direct observation of the behavior of individual DNA molecules, but act on length and timescales that are often too large and fast to observe underlying DNA and DNA–protein dynamics. Acting on length and timescales that complement single-molecule experiments, molecular dynamics simulations provide a high-resolution glimpse into the mechanics of a biomolecular world. Here, several simulation studies are presented, each of which quantified one or more properties of DNA. Specifically, the repulsive forces between parallel duplex DNA molecules were measured; the short-ranged, attractive end-to-end stacking energy was obtained; a single-stranded DNA model was developed that reproduced experimental measurements of its extension upon applied force; and finally the nature of single-stranded DNA binding to a single-stranded DNA binding protein was investigated. These works represent important steps towards larger simulations of more biologically complete DNA–protein systems.
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Aksimentiev%2C%20Aleksei%22%29&pagesize-30">Aksimentiev, Aleksei (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Schulten%2C%20Klaus%22%29&pagesize-30">Schulten, Klaus (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Aksimentiev%2C%20Aleksei%22%29&pagesize-30">Aksimentiev, Aleksei (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Stack%2C%20John%20D.%22%29&pagesize-30">Stack, John D. (committee member).
Subjects/Keywords: Deoxyribonucleic Acid (DNA); all-atom molecular dynamics; coarse-grained molecular dynamics; single-stranded DNA binding protein
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Maffeo, C. (2015). Quantitative all-atom and coarse-grained molecular dynamics simulation studies of DNA. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/72871
Chicago Manual of Style (16th Edition):
Maffeo, Christopher. “Quantitative all-atom and coarse-grained molecular dynamics simulation studies of DNA.” 2015. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/72871.
MLA Handbook (7th Edition):
Maffeo, Christopher. “Quantitative all-atom and coarse-grained molecular dynamics simulation studies of DNA.” 2015. Web. 01 Mar 2021.
Vancouver:
Maffeo C. Quantitative all-atom and coarse-grained molecular dynamics simulation studies of DNA. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2015. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/72871.
Council of Science Editors:
Maffeo C. Quantitative all-atom and coarse-grained molecular dynamics simulation studies of DNA. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2015. Available from: http://hdl.handle.net/2142/72871
University of Illinois – Urbana-Champaign
17.
Guzman Sanchez, Irisbel.
Kinetics and thermodynamics of protein-RNA interactions and protein folding in vitro and in cells.
Degree: PhD, Biochemistry, 2015, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/78610
► Protein-RNA interactions and protein folding are critical subjects in biochemistry, because of their significance during the formation of active complexes and signaling pathways. Regardless of…
(more)
▼ Protein-RNA interactions and protein folding are critical subjects in biochemistry, because of their significance during the formation of active complexes and signaling pathways. Regardless of the substantial amount of studies in the fields of protein-RNA interactions and protein folding, little is known about the stability and kinetics of these in the cell. This doctoral dissertation aims to advance the understanding of protein-RNA interactions and protein folding inside cells through comparative in vitro studies, utilizing U1A-SL2 RNA complex and PGK/VlsE proteins as model systems, respectively. For the protein-RNA studies, dynamics experiments of one positive charged mutant of the spliceosomal U1A protein, the golden model for the RNA Recognition Motif (RRM), reveled a conformational transition for the protein only. Also, U1A-SL2 RNA dissociation kinetics studies with U1A positive charged mutants supported the previously proposed two-step dissociation pathway and demonstrated the importance of positive charge residues. The U1A-SL2 was also investigated in macromolecular crowded buffers were its binding affinity increased. It was also studied inside mammalian cells were it localized in the nucleus and its binding affinity decreased. For the protein folding studies, the extracellular VlsE antigen was found to be destabilized inside mammalian cells opposed to the intracellular PGK enzyme.
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Gruebele%2C%20Martin%22%29&pagesize-30">Gruebele, Martin (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Martin%20Gruebele%22%29&pagesize-30">Martin Gruebele (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Gennis%2C%20Robert%22%29&pagesize-30">Gennis, Robert (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Ceman%2C%20Stephanie%20S.%22%29&pagesize-30">Ceman, Stephanie S. (committee member).
Subjects/Keywords: Protein-RNA interactions; protein folding; fluorescence; fluorescence resonance energy transfer (FRET); fast relaxation imagining (FREI); temperature jump
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
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APA (6th Edition):
Guzman Sanchez, I. (2015). Kinetics and thermodynamics of protein-RNA interactions and protein folding in vitro and in cells. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/78610
Chicago Manual of Style (16th Edition):
Guzman Sanchez, Irisbel. “Kinetics and thermodynamics of protein-RNA interactions and protein folding in vitro and in cells.” 2015. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/78610.
MLA Handbook (7th Edition):
Guzman Sanchez, Irisbel. “Kinetics and thermodynamics of protein-RNA interactions and protein folding in vitro and in cells.” 2015. Web. 01 Mar 2021.
Vancouver:
Guzman Sanchez I. Kinetics and thermodynamics of protein-RNA interactions and protein folding in vitro and in cells. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2015. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/78610.
Council of Science Editors:
Guzman Sanchez I. Kinetics and thermodynamics of protein-RNA interactions and protein folding in vitro and in cells. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2015. Available from: http://hdl.handle.net/2142/78610
University of Illinois – Urbana-Champaign
18.
Suksombat, Sukrit.
Binding configurations of single-stranded DNA binding protein and their influence on DNA recombinase.
Degree: PhD, Physics, 2015, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/78654
► DNA inside a cell is continuously damaged through multiple mechanisms including environmental exposure to radiation, chemical agents, or UV light. Certain products of the cell's…
(more)
▼ DNA inside a cell is continuously damaged through multiple mechanisms including environmental exposure to radiation, chemical agents, or UV light. Certain products of the cell's own metabolism, such as reactive oxygen species, can also damage the DNA. In the worst-case scenario, this damage results in double-stranded DNA (dsDNA) breaks. Double-stranded DNA breaks are lethal, and difficult to repair, with potential complications from genome rearrangement. To prevent this genetic instability, a cell can utilize a homologous chromosome as a template to accurately repair DSBs. This process is called homologous recombination.
Homologous recombination begins when an enzyme complex binds to a blunt end of a dsDNA break. The complex unzips the dsDNA through its helicase activity, and simultaneously cleaves the newly-generated 5' end of the ssDNA. This process leaves the remaining ssDNA strand exposed to the surrounding environment and prone to nucleolytic and chemical attacks. Cells have evolved single-stranded DNA binding (SSB) proteins to wrap and protect this ssDNA. In E. coli, SSB is known to wrap ssDNA in a variety of binding configurations, or modes. Three different binding modes, (SSB)65, (SSB)56, and (SSB)35, which wraps 65, 56, and 35 nucleotides (nt) respectively, have been observed in vitro Previous studies have suggested that SSB binding in different modes may exhibit different levels of binding cooperativity. SSBs in the (SSB)65 binding mode form isolated clusters (limited cooperativity), while SSBs in the (SSB)35 binding mode form long filaments (unlimited cooperativity). These different levels of binding cooperativity have been proposed to be used selectively in different DNA metabolic processes, including DNA replication, recombination, and repair.
In homologous recombination, recombinase RecA must bind and form nucleoprotein filaments on the ssDNA, in direct competition with SSB. Prior studies have shown that RecA is capable of forming filaments on ssDNA wrapped by SSBs in the (SSB)65 binding mode, but filament formation on ssDNA wrapped by SSBs in the (SSB)35 binding mode is inhibited. Recent single-molecule studies have been conducted to investigate this competitive process, but the detailed mechanisms remain unclear.
Here, we use high-resolution optical tweezers with simultaneous fluorescence microscopy to observe directly the activity of ssDNA-SSB, ssDNA-RecA, and ssDNA-SSB-RecA complexes under tension, and characterize their mechanical properties. The instrument allows us to simultaneously probe and visualize the interactions of RecA and SSB with ssDNA in real time and with nanometer resolution.
We confirm that individuals SSBs bind and compact ssDNA in discrete modes. Under low tension (1-3 pN), a single SSB wraps ssDNA in the (SSB)65 or (SSB)56 binding mode. At higher tension (4-8 pN), SSB exhibits transient wrapping-unwrapping, switching between the (SSB)56, (SSB)35, and (SSB)17 wrapping modes. When multiple SSBs are present on the ssDNA, the SSBs form isolated clusters in those solution…
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Chemla%2C%20Yann%20R.%22%29&pagesize-30">Chemla, Yann R. (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Aksimentiev%2C%20Aleksei%22%29&pagesize-30">Aksimentiev, Aleksei (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Myong%2C%20Su-A%22%29&pagesize-30">Myong, Su-A (committee member).
Subjects/Keywords: Optical Tweezers; Single-stranded DNA Binding Protein; Single-stranded DNA binding (SSB); RecA; Deoxyribonucleic Acid (DNA); Single-Molecule; Optical Traps
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
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APA (6th Edition):
Suksombat, S. (2015). Binding configurations of single-stranded DNA binding protein and their influence on DNA recombinase. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/78654
Chicago Manual of Style (16th Edition):
Suksombat, Sukrit. “Binding configurations of single-stranded DNA binding protein and their influence on DNA recombinase.” 2015. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/78654.
MLA Handbook (7th Edition):
Suksombat, Sukrit. “Binding configurations of single-stranded DNA binding protein and their influence on DNA recombinase.” 2015. Web. 01 Mar 2021.
Vancouver:
Suksombat S. Binding configurations of single-stranded DNA binding protein and their influence on DNA recombinase. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2015. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/78654.
Council of Science Editors:
Suksombat S. Binding configurations of single-stranded DNA binding protein and their influence on DNA recombinase. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2015. Available from: http://hdl.handle.net/2142/78654
University of Illinois – Urbana-Champaign
19.
Li, Yang.
Algorithms for analyzing complex structural variations in cancer genomes.
Degree: PhD, Bioengineering, 2015, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/88190
► Analysis of somatic alterations in cancer genomes has been accelerated through the rapid growth of the quantity, quality and depth of data generated by next-generation…
(more)
▼ Analysis of somatic alterations in cancer genomes has been accelerated through the rapid growth of the quantity, quality and depth of data generated by next-generation sequencing (NGS). Previously most of cancer genome studies were focusing on single nucleotide variations (SNVs), small insertions and deletions (INDELs), or somatic copy number alterations (SCNAs). Recently, there is a paradigm shift in the cancer genome study that more efforts have been devoted to characterizing large scale structural variations (SVs) in various cancer genomes. However, there are still pressing needs for designing specific computational algorithms to tackle the challenges caused by the complexity of cancer genomes.
The first part of my thesis is developing a novel computational method called Weaver, which takes whole genome sequencing (WGS) alignment as core input and generates a precise rearrangement map for cancer genomes. Weaver identifies SVs with base-pair resolution and applies a probabilistic graphical model to simultaneously quantify allele specific copy number of SVs (ASCNS) and genomic regions (ASCNG). Through evaluation on simulated datasets with different parameter settings, Weaver was demonstrated to be highly accurate and be able to significantly refine the analysis of complex cancer genomes.
The second part of this study is applying Weaver on two widely used cancer cell lines: MCF-7 and HeLa. For both cell lines, we generated base-pair resolution ASCNS and ASCNG for the first time. The detailed characterization of genomes for MCF-7 and HeLa may serve as valuable resource for future studies based on these two cell lines, by replacing reference genome with cancer specific genomes. We have found that allele specific expression can be explained by the profiled ASCNG for both cell lines. We have also discovered that a large portion of promoter-promoter interactions, detected by ChIA-PET, are found to be formed by distal genomic regions linked to be adjacent by somatic translocations in MCF-7 genome, showing that phased SVs analysis by Weaver has enabled the analysis of interaction between genomic rearrangements and long-range gene regulation at much broader scale.
The last part of this thesis is applying Weaver on large-scale primary tumor data, com- posed by 600 TGCA WGS samples. To our knowledge, this is the largest whole genome SV and base-pair resolution ASCNG analysis for primary cancer genomes to date. We analyzed two mechanisms, breakage-fusion-bridge (BFB) and tandem duplication (TD), for recurrent focal amplifications and found different frequently focal amplified regions have different enrichment of specific tumor types. We proposed a new pan-cancer classification method, for the first time utilizing SV pattern, that categorizes 600 TCGA samples across 17 tumor types into five subtypes with potential clinical relevance. Our pan-cancer classification has the potential of prognostic assessment for future patients regardless of their tumor types.
In order to gain knowledge on the landscape of cancer genome…
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Ma%2C%20Jian%22%29&pagesize-30">Ma, Jian (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Ma%2C%20Jian%22%29&pagesize-30">Ma, Jian (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Song%2C%20Jun%22%29&pagesize-30">Song, Jun (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Sinha%2C%20Saurabh%22%29&pagesize-30">Sinha, Saurabh (committee member).
Subjects/Keywords: allele specific copy number alteration; structural variation; tandem duplication; breakage-fusion-bridge; chromothripsis; markov chain model; cancer genome sequencing; MCF-7
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APA ·
Chicago ·
MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Li, Y. (2015). Algorithms for analyzing complex structural variations in cancer genomes. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/88190
Chicago Manual of Style (16th Edition):
Li, Yang. “Algorithms for analyzing complex structural variations in cancer genomes.” 2015. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/88190.
MLA Handbook (7th Edition):
Li, Yang. “Algorithms for analyzing complex structural variations in cancer genomes.” 2015. Web. 01 Mar 2021.
Vancouver:
Li Y. Algorithms for analyzing complex structural variations in cancer genomes. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2015. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/88190.
Council of Science Editors:
Li Y. Algorithms for analyzing complex structural variations in cancer genomes. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2015. Available from: http://hdl.handle.net/2142/88190
University of Illinois – Urbana-Champaign
20.
Blehm, Benjamin.
Force studies of intracellular transport.
Degree: PhD, 0240, 2012, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/32029
► Cytoskeletal motors like kinesin-1 and dynein are necessary for intracellular transport and a variety of other functions in the cell. They have been well characterized…
(more)
▼ Cytoskeletal motors like kinesin-1 and dynein are necessary for intracellular transport and a variety of other functions in the cell. They have been well characterized in simplified, single-motor, in vitro systems, but less is known about their mechanical properties in vivo, in a more complex, multi-motor environment. In order to better study these properties and their impact on intracellular transport, we have built an optical trap to implement a recently developed theoretical technique which allows us to calibrate and measure forces in a living cell and other viscoelastic environments. We have found that lipid droplets in A549 cells and phagocytosed beads in Dictyostelium cells typically have 1 active plus-end directed motor and 1 active minus-end directed motor. Also, the plus-end motor’s stall force appears to be lower (2-3 pN) than kinesin-1’s in vitro stall force (5-7 pN), while the minus-end motor’s stall force (2 pN) is slightly higher but similar to many in vitro measurements of dynein’s stall force. Stall force measurements made in vitro by measuring the stall force of beads with both kinesin and dynein attached give results similar to those in vivo, supporting a synergistic transport model, in which dynein remains attached to the microtubule at all times, being dragged behind the kinesin and reducing its stall force when the cargo is moving in the plus direction, and being the sole active motor when the cargo is moving in the negative direction.
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Selvin%2C%20Paul%20R.%22%29&pagesize-30">Selvin, Paul R. (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Selvin%2C%20Paul%20R.%22%29&pagesize-30">Selvin, Paul R. (committee member),
Champaign%22%20%2Bcontributor%3A%28%22DeMarco%2C%20Brian%20L.%22%29&pagesize-30">DeMarco, Brian L. (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Oono%2C%20Yoshitsugu%22%29&pagesize-30">Oono, Yoshitsugu (committee member).
Subjects/Keywords: Optical Trap; Tweezers; Stall Force; Intracellular transport; Organelle transport; Kinesin; dynein; synergistic model; motor coordination
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APA (6th Edition):
Blehm, B. (2012). Force studies of intracellular transport. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/32029
Chicago Manual of Style (16th Edition):
Blehm, Benjamin. “Force studies of intracellular transport.” 2012. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/32029.
MLA Handbook (7th Edition):
Blehm, Benjamin. “Force studies of intracellular transport.” 2012. Web. 01 Mar 2021.
Vancouver:
Blehm B. Force studies of intracellular transport. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2012. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/32029.
Council of Science Editors:
Blehm B. Force studies of intracellular transport. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2012. Available from: http://hdl.handle.net/2142/32029
University of Illinois – Urbana-Champaign
21.
Khafizov, Rustem.
Single molecule force spectroscopy of single stranded DNA binding protein and Rep helicase.
Degree: PhD, 0240, 2012, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/34441
► Single-stranded DNA binding (SSB) proteins are essential accessory proteins that protect single-stranded DNA (ssDNA) during genome maintenance. Escherichia coli SSB is a prototypical homo-tetrameric SSB…
(more)
▼ Single-stranded DNA binding (SSB) proteins are essential accessory proteins that protect single-stranded DNA (ssDNA) during genome maintenance. Escherichia coli SSB is a prototypical homo-tetrameric SSB protein that can wrap up to 65 nucleotides of ssDNA in one of its binding modes. Here we present mechanical studies of E. coli SSB bound to ssDNA using high-resolution optical tweezers. This method allows us to probe the interaction of individual SSBs to ssDNA in real time, with nanometer resolution. By detecting directly the wrapping of ssDNA by a single protein, we are able to characterize the thermodynamics and kinetics of nucleoprotein complex formation. Mechanical pulling of ssDNA in the presence of SSB reveals that the protein condenses ssDNA in the force range 0-10pN and that tension can be used to modulate the ssDNA wrapping state of SSB. Measurements of SSB kinetics indicate that SSB-ssDNA complex formation occurs in a two-step process consisting of a diffusion-limited binding step in which the protein associates weakly with its substrate, followed by a fast wrapping step in which ssDNA is condensed. We also quantify how tension modulates the ssDNA wrapping state of SSB, revealing features of the energy landscape for SSB-ssDNA interactions. Lastly, we carried out measurements of SSB interaction with long ssDNA binding substrate as a function of mechanical force. The data indicate that SSBs bound to longer stretches of ssDNA bound much tighter, probably due to nucleoprotein filament formation. And we have evidence that SSB can bind to ssDNA in intermediate wrapping states and are transiently wrapping and unwrapping from their substrates.
In addition we present for the first time study on the conformational control of Rep helicase using an optical trap. We found that crosslinking-mediated conformational arrest of a dynamic subdomain in so-called “closed” orientation converted the Superfamily I (SFI) helicase, Rep, from a very poor DNA helicase into a powerful motor protein with a highly processive DNA unwinding activity. In contrast, the wild type Rep helicase cannot efficiently unwind DNA over 18 bp in vitro. A single Rep-X (cross-linked Rep) molecule can processively unwind DNA up to 4 kbp and generate forces in excess of 40 pN, making it the most powerful helicase known. The same modification on the related PcrA helicase produced the same activity increase characteristics strengthening the possibility of widespread application of this conformational control technique. Thus our results directly answer the question of the role of different conformations observed in SF-I helicase crystal structures and offer a mechanism for how partner proteins in vivo may regulate helicase function via conformational control.
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Chemla%2C%20Yann%20R.%22%29&pagesize-30">Chemla, Yann R. (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Chemla%2C%20Yann%20R.%22%29&pagesize-30">Chemla, Yann R. (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Stack%2C%20John%20D.%22%29&pagesize-30">Stack, John D. (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Oono%2C%20Yoshitsugu%22%29&pagesize-30">Oono, Yoshitsugu (committee member).
Subjects/Keywords: Optical Trap; Single-stranded DNA binding protein; Single-stranded DNA binding (SSB); Rep helicase; single molecule
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APA (6th Edition):
Khafizov, R. (2012). Single molecule force spectroscopy of single stranded DNA binding protein and Rep helicase. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/34441
Chicago Manual of Style (16th Edition):
Khafizov, Rustem. “Single molecule force spectroscopy of single stranded DNA binding protein and Rep helicase.” 2012. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/34441.
MLA Handbook (7th Edition):
Khafizov, Rustem. “Single molecule force spectroscopy of single stranded DNA binding protein and Rep helicase.” 2012. Web. 01 Mar 2021.
Vancouver:
Khafizov R. Single molecule force spectroscopy of single stranded DNA binding protein and Rep helicase. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2012. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/34441.
Council of Science Editors:
Khafizov R. Single molecule force spectroscopy of single stranded DNA binding protein and Rep helicase. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2012. Available from: http://hdl.handle.net/2142/34441
University of Illinois – Urbana-Champaign
22.
Tsang, Ying-Hung N.
Regulation of RUNX3 in gastric and breast cancer.
Degree: PhD, 0318, 2012, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/29531
► RUNX3 is a transcription factor that is ubiquitously expressed in different tissues and has been shown to have diverse functions in many developmental procedures. Recently…
(more)
▼ RUNX3 is a transcription factor that is ubiquitously expressed in different tissues and has been shown to have diverse functions in many developmental procedures. Recently it has also been acknowledged that RUNX3 is involved as a tumor suppressor in many distinct cancers in different tissues. In this thesis, we will examine the regulation of this tumor suppressor in gastric cancer and breast cancer.
Chronic infection with cagA-positive Helicobacter pylori is the strongest risk factor for the development of gastric adenocarcinoma. The cagA gene product CagA is injected into gastric epithelial cells and disturbs cellular functions by physically interacting with and deregulating a variety of cellular signaling molecules. RUNX3 is expressed gastric epithelial tissues, and is frequently inactivated in gastric cancer. In the first part of the thesis, we showed that H. pylori infection inactivates the gastric tumor suppressor RUNX3 in a CagA-dependent manner. CagA directly associates with RUNX3 through a specific recognition of the PY motif of RUNX3 by a WW domain of CagA. Deletion of the WW domains of CagA or mutation of the PY motif in RUNX3 abolishes the ability of CagA to induce the ubiquitination and degradation of RUNX3, thereby extinguishing its ability to inhibit the transcriptional activation of RUNX3. This study identify RUNX3 as a novel cellular target of H. pylori CagA and also reveal a mechanism by which CagA functions as an oncoprotein by blocking the activity of gastric tumor suppressor RUNX3.
RUNX3 has also been known to be inactivated in breast cancer through dual mechanism of cytoplasmic mislocalization as well as promoter hypermethylation. Recent studies in our lab have shown that RUNX3 knockout mice have an approximate 20% increased chance of
ii
developing breast cancer compared to WT mice. At the same time, MCF7 cells expressing RUNX3 resulted in smaller tumor growth in a tumorigenicity assay compared to MCF7 cells expressing a control vector, further affirming RUNX3’s importance as a tumor suppressor in breast cancer. Pin1 is an isomerase that is over-expressed in human breast cancer. Pin1 specifically isomerizes only the Ser/Thr-Pro bonds in certain proteins, which allows it to act as a molecular switch controlling protein functions. In the second part of this thesis, we discuss the findings that Pin1 interacts specifically with tumor suppressor protein RUNX3 through 4 separate phosphorylated Ser/Thr-Pro motifs on the RUNX3 protein. Through this interaction with Pin1, the ubiquitination of RUNX3 is markedly enhanced, resulting in decreased protein stability. RUNX3 is therefore targeted to the 26S proteasome for degradation in the presence of Pin1. Our data shows a novel pathway through which tumor suppressor protein RUNX3 can be inactivated and hence regulated in breast tissues by Pin1.
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Chen%2C%20Lin-Feng%22%29&pagesize-30">Chen, Lin-Feng (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Chen%2C%20Lin-Feng%22%29&pagesize-30">Chen, Lin-Feng (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Blanke%2C%20Steven%20R.%22%29&pagesize-30">Blanke, Steven R. (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Fratti%2C%20Rutilio%20A.%22%29&pagesize-30">Fratti, Rutilio A. (committee member).
Subjects/Keywords: Runt-related transcription factor 3 (RUNX3); cytotoxin-associated gene A (CagA); Pin1; Gastric cancer; Breast cancer
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APA ·
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APA (6th Edition):
Tsang, Y. N. (2012). Regulation of RUNX3 in gastric and breast cancer. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/29531
Chicago Manual of Style (16th Edition):
Tsang, Ying-Hung N. “Regulation of RUNX3 in gastric and breast cancer.” 2012. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/29531.
MLA Handbook (7th Edition):
Tsang, Ying-Hung N. “Regulation of RUNX3 in gastric and breast cancer.” 2012. Web. 01 Mar 2021.
Vancouver:
Tsang YN. Regulation of RUNX3 in gastric and breast cancer. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2012. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/29531.
Council of Science Editors:
Tsang YN. Regulation of RUNX3 in gastric and breast cancer. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2012. Available from: http://hdl.handle.net/2142/29531
University of Illinois – Urbana-Champaign
23.
He, Ying.
Fluorescence resonance energy transfer study of the global folding of functional DNAs and electrohydrodynamic printing of protein arrays.
Degree: PhD, 0130, 2012, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/29571
► Fluorescence resonance energy transfer (FRET) is used to monitor the metal ion dependent conformational change of the UO22+ specific DNAzyme 39E. Inactive metal ions, Mg2+…
(more)
▼ Fluorescence resonance energy transfer (FRET) is used to monitor the metal ion dependent conformational change of the UO22+ specific DNAzyme 39E. Inactive metal ions, Mg2+ and Zn2+ are found capable of inducing folding between two pairs of stems while the active ions UO22+ and Pb2+ are not able to bring about the folding in any pairs of the stems. By correlating the enzymatic cleavage results, the Mg2+ and Zn2+ induced folding exerts a counter-productive effect
on the activities. This result contradicts with the normal productive role that Mg2+ plays in the activation of ribozyme functions. At reduced ionic strength, even UO22+ and Pb2+ can induce folding due to the nonspecific electrostatic interaction from their divalent metal ion nature. And the progression of Mg2+ and Na+ concentration leads to an inactive-active-inactive transition in
the DNAzyme function. Single molecule FRET is also used to obtain kinetics for low UO22+
concentration at reduced imaging buffer strength. Together with the biochemical characterization
for the catalytic cores and optimal working conditions, a thorough understanding of this
DNAzyme 39E is obtained, which might be helpful in the design of sensitive and selective
DNAzymes.
FRET is also used to gain step-wise kinetic information of adenosine aptamer structure-switching sensor. The kinetic information for individual steps (folding and releasing) obtained by
monitoring FRET process between fluorophores labeled at several positions of the aptamer
structure switching sensor provides direct evidence for sequential occurrences, as predicted by the structure switching principle. The information obtained here will facilitate sensors designed based
on the structure-switching principle.
Electrohydrodynamic jet (E-jet) printing is applied to the protein microarray field. With the development of multi-nozzle printing system, both single-protein array and multiple-protein
arrays are successfully demonstrated. Several proteins, such as streptavidin, Green fluorescence protein (GFP), mCherry (a red fluorescence protein), et.al, have been printed, proving E-jet printing is generally applicable to many types of proteins. The printed streptavidin maintains its
binding character with biotin, showing the printed streptavidin is still structurally intact and
functionally active. What???s more, this printing technology has also been employed to immunology application. Immunoglobulin (Ig) G from several animal species are printed by E-jet printing, and their binding specificities to corresponding secondary antibody, anti IgG, are maintained. To prove this technique suitable for more practical applications, an inch-sized array with well controlled feature details has been completely in a short period of time. Overall, this technique will be a promising candidate for future protein microarray fabrication method.
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Lu%2C%20Yi%22%29&pagesize-30">Lu, Yi (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Rogers%2C%20John%20A.%22%29&pagesize-30">Rogers, John A. (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Braun%2C%20Paul%20V.%22%29&pagesize-30">Braun, Paul V. (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Cheng%2C%20Jianjun%22%29&pagesize-30">Cheng, Jianjun (committee member).
Subjects/Keywords: Functional DNA; Fluorescence resonance energy transfer (FRET); Protein microarray; Deoxyribonucleic acid (DNA)
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
He, Y. (2012). Fluorescence resonance energy transfer study of the global folding of functional DNAs and electrohydrodynamic printing of protein arrays. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/29571
Chicago Manual of Style (16th Edition):
He, Ying. “Fluorescence resonance energy transfer study of the global folding of functional DNAs and electrohydrodynamic printing of protein arrays.” 2012. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/29571.
MLA Handbook (7th Edition):
He, Ying. “Fluorescence resonance energy transfer study of the global folding of functional DNAs and electrohydrodynamic printing of protein arrays.” 2012. Web. 01 Mar 2021.
Vancouver:
He Y. Fluorescence resonance energy transfer study of the global folding of functional DNAs and electrohydrodynamic printing of protein arrays. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2012. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/29571.
Council of Science Editors:
He Y. Fluorescence resonance energy transfer study of the global folding of functional DNAs and electrohydrodynamic printing of protein arrays. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2012. Available from: http://hdl.handle.net/2142/29571
University of Illinois – Urbana-Champaign
24.
Park, Seongjin.
Cellular responses to external threats probed by super-resolution microscopy.
Degree: PhD, Physics, 2015, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/89208
► Fluorescence microscopy has been an essential tool in biology. However, its imaging resolution has been limited around >200 nm in lateral dimensions, and >500 nm…
(more)
▼ Fluorescence microscopy has been an essential tool in biology. However, its imaging resolution has been limited around >200 nm in lateral dimensions, and >500 nm in axial dimension, leaving many biological structures too small to study. However the recent developments of several super-resolution techniques, this limit has been overcome. Here we present our approach to this matter, using our three-dimensional (3D) multicolor super-resolved single fluorophore microscopy. Especially we report imaging of a near-IR dye for imaging thick and dense structures for multicolor colocalization studies. Then we present three applications of the super-resolution imaging technique to the following three biological systems.
Viral infection in mammalian cells triggers the immune response, a cascade of antiviral signaling proteins. For double stranded RNA (dsRNA) virus, the innate immune response starts from retinoic acid-inducible gene-I (RIG-I) protein which is a dsRNA sensor. For certain types of viruses, RIG-I localizes in cytoplasmic granular aggregates called as antiviral granules (AVGs). We infected/transfected cells by influenza virus lacking NS1 (IAVΔNS1) or polyinosinic:polycytidylic acid (poly I:C) to induce AVGs. We found in AVGs, RIG-I forms clusters of around 110nm in diameter. Then we treated intact cells with various stress conditions to from stress granules (SGs) and could also observe RIG-I clusters in SGs. RIG-I was also clustered in intact cells, but the clustering percentage and diameter were small than in AVGs or SGs, so we conclude that the intrinsically clustered RIG-I relocalizes into granular structures upon external stimuli, and its clustering is enhanced. To verify RIG-I clustering, we imaged TIAR, a marker for SGs, which showed much less clustering. Also we conducted various tests on our clustering algorithm, as well as structural illumination microscopy (SIM) imaging that all support the idea of the clustering of RIG-I.
In bacterial cells, upon vast global DNA damage, an error-prone DNA repair response called SOS response occurs. SOS response is initiated by RecA, a protein essential for maintenance of DNA. It was reported that RecA forms a bundle to connect uncut and cut locus of in the event of double strand break, supporting the idea that RecA mediates homology search. We report that RecA also forms bundles in the SOS response, and these bundles are ribbon-like structures, i.e. flat at one side and wide at the other side, hypothetically wrapping around DNA damaged sites.
The conventional approach of applying broad-spectrum antibiotics to treat bacterial infections contributes to the emerging of antibiotic resistance. To cope with this, species specific and narrow-spectrum antibiotics draw attention. Plantazolicin (PZN) is a natural antibiotic that is highly specific against B. anthracis which is the agent of anthrax and a category A priority pathogen, but the mechanism of how PZN kills B. anthracis has been unknown. Recent investigation showed PZN depolarizes B. anthracis membrane, and it…
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">
Ha,
Taekjip (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Selvin%2C%20Paul%20R%22%29&pagesize-30">Selvin, Paul R (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Schroeder%2C%20Charles%20M%22%29&pagesize-30">Schroeder, Charles M (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Kuhlman%2C%20Thomas%20E%22%29&pagesize-30">Kuhlman, Thomas E (committee member).
Subjects/Keywords: super-resolution; microscopy; biophysics; fluorescence; Stochastic Optical Reconstruction Microscopy (STORM); Photoactivated localization microscopy (PALM); retinoic acid-inducible gene-I (RIG-I); antiviral; SOS; RecA; Plantazolicin; Plantazolicin (PZN); influenza; nonstructural one (ns1); T-cell restricted intracellular antigen-related protein (TIAR); stress granule; antiviral granules (AVG); stress granules (SG); structural illumination microscopy (SIM); structural illumination; deoxyribonucleic acid (DNA); virus; antibiotics; resolution limit; optical microscopy
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APA ·
Chicago ·
MLA ·
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CSE |
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to Zotero / EndNote / Reference
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APA (6th Edition):
Park, S. (2015). Cellular responses to external threats probed by super-resolution microscopy. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/89208
Chicago Manual of Style (16th Edition):
Park, Seongjin. “Cellular responses to external threats probed by super-resolution microscopy.” 2015. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/89208.
MLA Handbook (7th Edition):
Park, Seongjin. “Cellular responses to external threats probed by super-resolution microscopy.” 2015. Web. 01 Mar 2021.
Vancouver:
Park S. Cellular responses to external threats probed by super-resolution microscopy. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2015. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/89208.
Council of Science Editors:
Park S. Cellular responses to external threats probed by super-resolution microscopy. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2015. Available from: http://hdl.handle.net/2142/89208
University of Illinois – Urbana-Champaign
25.
Earnest, Tyler M.
Stochastic and physical modeling of fundamental biological processes.
Degree: PhD, Physics, 2016, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/95262
► Modeling is a necessary tool to understand the large volumes of data generated from quantitative experiments on biological systems. It combines our knowledge of a…
(more)
▼ Modeling is a necessary tool to understand the large volumes of data generated from quantitative experiments on biological systems. It combines our knowledge of a phenomenon into a succinct mathematical or computational description. In this dissertation, we first describe briefly two applications of modeling in biophysics: loading of the replication clamp into the replisome in the archaeon Methanosarcina acetivorans and genome packing initiation during the self-assembly of the T4 bacteriophage. We then describe in detail two systems: an improved model of the lac genetic switch which includes DNA looping in its gene regulation mechanism, and a spatially resolved, whole-cell model of ribosome biogenesis in Escherichia coli, which we then extend to include cell growth and replication of its genome.
For the first system, conditions and parameters affecting the range of bistability of the lac genetic switch in E. coli are examined for a model which includes DNA looping interactions with the lac repressor and a lactose analog. This stochastic gene-mRNA-protein model of the lac switch describes DNA looping using a third transcriptional state. We exploit the fast bursting dynamics of mRNA by combining a novel geometric burst approximation with the Finite State Projection method. This limits the number of protein/mRNA states, allowing for an accelerated search of the model's parameter space. We evaluate how the addition of the third transcriptional state changes the bistability properties of the model and find a critical region of parameter space where the phenotypic switching occurs in a range seen in single molecule fluorescence studies. Stochastic simulations show induction in the looping model is preceded by a rare complete dissociation of the loop followed by an immediate burst of mRNA rather than a slower build up of mRNA as in the two-state model. The overall effect of the looped state is to allow for faster switching times while at the same time further differentiating the uninduced and induced phenotypes. Furthermore, the kinetic parameters are consistent with free energies derived from thermodynamic studies suggesting that this minimal model of DNA looping could have a broader range of application.
For the second system, we study the biogenesis of the ribosome. Central to all life is the assembly of the ribosome: a coordinated process involving the hierarchical association of ribosomal protein to the RNAs forming the small and large ribosomal subunits. The process is further complicated by effects arising from the intracellular heterogeneous environment and the location of ribosomal operons within the cell. We provide a simplified model of ribosome biogenesis in slow growing E. coli. Kinetic models of in vitro small subunit reconstitution at the level of individual ribosomal protein to ribosomal RNA interactions are developed for two temperature regimes. The model at low temperatures predicts the existence of a novel 5’-3’-central assembly pathway, which we investigate further using molecular dynamics. The high…
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Luthey-Schulten%2C%20Zaida%22%29&pagesize-30">Luthey-Schulten, Zaida (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Chemla%2C%20Yann%20R%22%29&pagesize-30">Chemla, Yann R (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Kuhlman%2C%20Thomas%20E%22%29&pagesize-30">Kuhlman, Thomas E (committee member).
Subjects/Keywords: ribosome; ribosome biogenesis; stochastic modeling; lac operon; lac switch; chemical master equation; reaction-diffusion master equation; lattice microbes
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APA ·
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APA (6th Edition):
Earnest, T. M. (2016). Stochastic and physical modeling of fundamental biological processes. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/95262
Chicago Manual of Style (16th Edition):
Earnest, Tyler M. “Stochastic and physical modeling of fundamental biological processes.” 2016. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/95262.
MLA Handbook (7th Edition):
Earnest, Tyler M. “Stochastic and physical modeling of fundamental biological processes.” 2016. Web. 01 Mar 2021.
Vancouver:
Earnest TM. Stochastic and physical modeling of fundamental biological processes. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2016. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/95262.
Council of Science Editors:
Earnest TM. Stochastic and physical modeling of fundamental biological processes. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2016. Available from: http://hdl.handle.net/2142/95262
University of Illinois – Urbana-Champaign
26.
Zhang, Jichuan.
Developing high-resolution methods to study DNA and RNA dynamics.
Degree: PhD, Materials Science & Engr, 2016, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/95570
► Nucleic acids, including DNA and RNA, are one of the most important biomacromolecules inside the cell. DNA stores genetic information, while RNA has more versatile…
(more)
▼ Nucleic acids, including DNA and RNA, are one of the most important biomacromolecules inside the cell. DNA stores genetic information, while RNA has more versatile roles including conveying and deciphering genetic information, catalyzing biological reactions, conducting post- transcriptional regulation, and even storing genetic information. To understand the functioning principles of cells and living organisms, it is very important to study DNA- and RNA-involved biochemical reactions as well as the molecular mechanisms behind them, which is quite challenging due to the small size and high metabolism rate of cells. Thanks to the technology advancement in the past several decades, we are able to develop research methods to achieve high-rate, high-resolution and high-throughput study on DNA and RNA. Here we developed several fluorescence-based high-resolution assays to study DNA and RNA dynamics in vitro and in vivo.
First, we established a single molecule FRET assay to study homodimeric single-stranded binding protein (Thermus thermophilus SSB) specifically binding and protecting single-stranded DNA during DNA metabolism. With the help of specifically designed DNA constructs and total internal reflection microscopy, we discovered that homodimeric SSB showed similar one- dimensional diffusion and salt-dependent binding mode transition behavior which was confirmed for intensively studied homotetrameric SSB, suggesting that those behaviors might be universal among SSB homologues from different organisms.
Second, we developed a live cell RNA labeling and imaging method based on an aptamer- fluorogen system called “Spinach”, which contains an RNA sequence that binds a fluorogen DFHBI and induces its fluorescence. We constructed a Spinach array with tandem Spinach repeats and it greatly enhanced the cellular fluorescence signal, and we could easily visualize mRNAs in the cell. We further characterized the Spinach RNA imaging method and found that either single Spinach or Spinach arrays do not affect RNA transcription, protein translation or RNA degradation. Therefore we proposed that aptamer/fluorogen imaging and aptamer array construction could be a generalizable strategy for high performance and low perturbation live cell RNA imaging.
Finally, we expanded the research to gene expression regulation. Our research target, sgrS, is a bacterial small RNA that regulates several target genes at post-transcriptional level in response to sugar-phosphate stress. It is known that sgrS anneals target transcripts via basepairing interaction with the guide of Hfq protein. Nevertheless, how individual nucleotides within sgrS sequence contribute to the regulation process is not clear. Here we utilized the recently developed Sort-Seq method combining fluorescence-activated cell sorting (FACS) and high-throughput DNA sequencing to study sequence-dependent sgrS regulation on its primary target, ptsG. We constructed a target-reporter system with ptsG 5’ UTR, which is responsible for sgrS annealing, fused to GFP, whose expression level is…
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">
Ha,
Taekjip (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Cheng%2C%20Jianjun%22%29&pagesize-30">Cheng, Jianjun (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Kilian%2C%20Kristopher%22%29&pagesize-30">Kilian, Kristopher (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Kuhlman%2C%20Thomas%20E%22%29&pagesize-30">Kuhlman, Thomas E (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Leal%2C%20Cecilia%22%29&pagesize-30">Leal, Cecilia (committee member).
Subjects/Keywords: single-molecule imaging; FRET; DNA-protein interaction; RNA labeling; bacterial small RNA; fluorescence-activated cell sorting; high-throughput sequencing
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APA (6th Edition):
Zhang, J. (2016). Developing high-resolution methods to study DNA and RNA dynamics. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/95570
Chicago Manual of Style (16th Edition):
Zhang, Jichuan. “Developing high-resolution methods to study DNA and RNA dynamics.” 2016. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/95570.
MLA Handbook (7th Edition):
Zhang, Jichuan. “Developing high-resolution methods to study DNA and RNA dynamics.” 2016. Web. 01 Mar 2021.
Vancouver:
Zhang J. Developing high-resolution methods to study DNA and RNA dynamics. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2016. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/95570.
Council of Science Editors:
Zhang J. Developing high-resolution methods to study DNA and RNA dynamics. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2016. Available from: http://hdl.handle.net/2142/95570
University of Illinois – Urbana-Champaign
27.
Shah, Syed Tahir Abbas.
Regulation of SNARE function during sperm capacitation to promote the acrosome reaction.
Degree: PhD, 0002, 2013, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/45667
► Infertility is a tragedy for millions of couples worldwide. According to the World Health Organization, the male factor alone accounts for over one third of…
(more)
▼ Infertility is a tragedy for millions of couples worldwide. According to the World Health Organization, the male factor alone accounts for over one third of total infertility. There are various causes of male infertility but defective acrosomal exocytosis is perhaps one of the biggest contributors towards male infertility. Unfortunately there is little information available on the most frequent molecular causes of the inability of sperm to undergo acrosomal exocytosis. In the current study, my goal was to develop a clearer understanding of how the acrosome reaction is regulated during capacitation so that the molecular defects that cause infertility can be identified. I hypothesized that capacitation alters sperm protein phosphorylation promoting soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation and movement into membrane rafts in preparation for the acrosome reaction. Two t-SNAREs and a v-SNARE form a trans-SNARE complex, which is required for exocytosis. The core complex along with other regulatory proteins exerts an inward force by zippering from the N-termini. Ultimately hundreds of fusion pores are formed and hybrid vesicles are released. To test this hypothesis, I incubated mouse sperm in a capacitating (dmKRBT) or non-capacitating (dmKRBT without BSA or without HCO3-) medium and detergent-extracted sperm protein. To assess phosphorylation of proteins in SNARE complexes, syntaxin and associated proteins were immunoprecipitated with a syntaxin antibody and subsequently separated by SDS-PAGE. Samples were not boiled prior to SDS-PAGE to maintain the integrity of the SNARE complex. To detect changes in total phosphorylation of syntaxin-containing (SNARE) complexes, immunoprecipitated complexes were separated by SDS-PAGE and stained with Pro-Q Diamond. I observed phosphoprotein staining in 75, 100, and 150 and 230 KD protein complexes. Immunoblotting with a syntaxin antibody demonstrated that each complex contained syntaxin and was presumably a SNARE complex. After 30 min of capacitation, overall phosphorylation of SNARE complexes was higher in the 75, 100 and 150 KD complexes. To detect tyrosine phosphorylation of syntaxin and associated proteins, the anti-syntaxin immunoprecipitates were blotted with a phosphotyrosine antibody. Tyrosine phosphorylation of SNARE complexes decreased after 15 min of capacitation time. These data demonstrate SNARE complex phosphorylation is a dynamic process during capacitation and suggest that phosphorylation may regulate SNARE complex formation during capacitation in preparation for the acrosome reaction. Movement of SNAREs into membrane rafts was assessed using a detergent-free sucrose density gradient centrifugation method followed by immunoblotting with syntaxin and synaptobrevin antibodies. I observed movement of syntaxin and synaptobrevin into rafts in sperm incubated with capacitating dmKRBT and was delayed in dmKRBT lacking bicarbonate. No movement of syntaxin and synaptobrevin in sperm incubated with non-capacitating dmKRBT or…
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Miller%2C%20David%20J.%22%29&pagesize-30">Miller, David J. (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Miller%2C%20David%20J.%22%29&pagesize-30">Miller, David J. (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Nowak%2C%20Romana%20A.%22%29&pagesize-30">Nowak, Romana A. (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Knox%2C%20Robert%20V.%22%29&pagesize-30">Knox, Robert V. (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Fratti%2C%20Rutilio%20A.%22%29&pagesize-30">Fratti, Rutilio A. (committee member).
Subjects/Keywords: Capacitation; Acrosome Reaction; Infertility; Fertilization; Membrane fusion; Phosphorylation; soluble N-ethylmaleimide-sensitive factor attachment protein receptor; Lipid rafts; SNARE Proteins
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APA (6th Edition):
Shah, S. T. A. (2013). Regulation of SNARE function during sperm capacitation to promote the acrosome reaction. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/45667
Chicago Manual of Style (16th Edition):
Shah, Syed Tahir Abbas. “Regulation of SNARE function during sperm capacitation to promote the acrosome reaction.” 2013. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/45667.
MLA Handbook (7th Edition):
Shah, Syed Tahir Abbas. “Regulation of SNARE function during sperm capacitation to promote the acrosome reaction.” 2013. Web. 01 Mar 2021.
Vancouver:
Shah STA. Regulation of SNARE function during sperm capacitation to promote the acrosome reaction. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2013. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/45667.
Council of Science Editors:
Shah STA. Regulation of SNARE function during sperm capacitation to promote the acrosome reaction. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2013. Available from: http://hdl.handle.net/2142/45667
University of Illinois – Urbana-Champaign
28.
Chen, Ke.
Evolution and assembly of the ribosome.
Degree: PhD, 0319, 2014, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/46886
► The ribosome is a large macromolecular assembly responsible for protein synthesis in all living cells. A typical bacterial ribosome consists of three ribosomal RNA (rRNA)…
(more)
▼ The ribosome is a large macromolecular assembly responsible for protein synthesis in all living cells. A typical bacterial ribosome consists of three ribosomal RNA (rRNA) molecules and approximately fifty ribosomal proteins (r-proteins), which are arranged into two subunits of unequal size and distinct function. The large subunit promotes formation of the peptide bond, and the small subunit enforces the recognition between the mRNA codons and the tRNA anticodons. With the availability of the ribosome crystal structure, it becomes clear that the two major functions, peptide bond formation and decoding, are performed within an entire RNA environment devoid of proteins. Combining with the fact that the majority of the ribosomal components are conserved across all three domains of life, it is believed that the ribosome has its origin deep in the RNA world before the last universal common ancestor (LUCA). Further evolutionary studies lead to the hypothesis that the evolution of the ribosome begins with a prototype ribozyme that catalyzes peptidyl-transferase reaction.
Structural and sequence analysis suggests that the small ribozyme capable of catalyzing formation of short peptides may still exist in the core of the modern ribosome. Accordingly, a proto-ribosome model is constructed computationally using RNA fragments near the peptidyl-transferase center (PTC), and is proven to be stable throughout the micro-second molecular dynamics (MD) simulations. The model is capable of incorporating freely diffusing substrates spontaneously into its binding site, and holds them in both pockets long enough to reach a transition intermediate favorable for peptide bond formation. This in silico designed proto-ribosome is then subjected to experimental investigations to test its ability to assemble and bind potential substrates in solution. The successful design of the proto-ribosome presents a possible scenario for the initial development of the early translation apparatus. The proto-ribosome coupled with the probable parallel evolution of ancient tRNAs might have driven the emergence of the oldest coded protein shortly afterwards.
The universally conserved r-protein S4 is likely an ancient protein due to its role in the initiation of the 30S assembly, control of the translational accuracy, and regulation of the conserved operon. However, the N-terminal domain of S4 is identified as a “molecular signature” that distinguishes between Bacteria and Archaea, and hence might be a newer addition to the protein. The presence of both an old and a new component in the same protein makes it an extremely interesting case to study for the ribosomal evolution. Therefore, we perform phylogenetic analysis of S4 in relation to a broad sharing of zinc/non-zinc binding sequence in the N-terminal domain of the protein, and study the scope of horizontal gene transfer (HGT) of S4 during bacterial evolution. The complex history presented for “core” protein S4 suggests the existence of a gene pool before the emergence of bacterial lineages and reflects…
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Luthey-Schulten%2C%20Zaida%20A.%22%29&pagesize-30">Luthey-Schulten, Zaida A. (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Martin%20Gruebele%22%29&pagesize-30">Martin Gruebele (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Luthey-Schulten%2C%20Zaida%20A.%22%29&pagesize-30">Luthey-Schulten, Zaida A. (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Schulten%2C%20Klaus%20J.%22%29&pagesize-30">Schulten, Klaus J. (committee member).
Subjects/Keywords: the RNA world; last universal common ancestor (LUCA); ribosomal evolution; ribosomal signature; ribosomal assembly; ribosomal RNA (rRNA); r-proteins; r-protein S4; RNA folding; protein binding; ligand binding; fly-casting mechanism; single-molecule Förster resonance energy transfer (smFRET); selective 20-hydroxyl acylation analyzed by primer extension (SHAPE); footprinting; molecular dynamics simulation; structure-based Go potential; micro-second simulation
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Chen, K. (2014). Evolution and assembly of the ribosome. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/46886
Chicago Manual of Style (16th Edition):
Chen, Ke. “Evolution and assembly of the ribosome.” 2014. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/46886.
MLA Handbook (7th Edition):
Chen, Ke. “Evolution and assembly of the ribosome.” 2014. Web. 01 Mar 2021.
Vancouver:
Chen K. Evolution and assembly of the ribosome. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2014. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/46886.
Council of Science Editors:
Chen K. Evolution and assembly of the ribosome. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2014. Available from: http://hdl.handle.net/2142/46886
University of Illinois – Urbana-Champaign
29.
Giri, Sumanprava.
Role of ORCA and ORC in chromatin organization and DNA replication.
Degree: PhD, Cell and Developmental Biology, 2016, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/90477
► In eukaryotes, Origin recognition complex (ORC) proteins establish the pre-replicative complex (pre-RC) at the origins and this is essential for the initiation of DNA replication.…
(more)
▼ In eukaryotes, Origin recognition complex (ORC) proteins establish the pre-replicative complex (pre-RC) at the origins and this is essential for the initiation of DNA replication. In human cells, ORC is a highly dynamic complex with many separate functions attributed to sub-complexes or individual subunits of ORC including heterochromatin organization, telomere and centromere function, centrosome duplication and cytokinesis.
Heterochromatic domains are enriched with repressive histone marks, including histone H3 lysine 9 methylation, written by lysine methyltransferases (KMTs). ORC along with the pre-RC protein Origin Recognition Complex-Associated (ORCA/LRWD1), preferentially localizes to heterochromatic regions in post-replicated cells. The role of ORCA and ORC in heterochromatin organization remained elusive. In Chapter II, I describe my efforts to understand the significance of ORCA-ORC’s association with heterochromatin. ORCA recognizes methylated H3K9 marks and interacts with repressive KMTs, including G9a/GLP and Suv39H1 in a chromatin context-dependent manner. Single-molecule pull-down assays demonstrate that ORCA-ORC and multiple H3K9 KMTs exist in a single complex and that ORCA stabilizes H3K9 KMT complex. Cells lacking ORCA show alterations in chromatin architecture, with significantly reduced H3K9 di- and tri-methylation at specific chromatin sites. Changes in heterochromatin structure due to loss of ORCA affects replication timing, preferentially at the late-replicating regions. I demonstrate that ORCA acts as a scaffold for the establishment of H3K9 KMT complex and its association and activity at specific chromatin sites is crucial for the organization of heterochromatin structure.
Heterochromatin mostly constitutes tightly packaged DNA, decorated with repressive histone marks, including histone H3 methylated at lysine 9, histone H4 methylated at lysine 20 and histone H3 methylated at lysine 27. Each of these marks is incorporated by specific histone lysine methyl transferases. While constitutive heterochromatin enriched with H3K9me3 and H4K20me3 occur within repetitive elements, including centromeres and telomeres, the facultative heterochromatin resides on the inactive X-chromosome and contains H3K27me3 mark. ORCA associates with constitutive and facultative heterochromatin in human cells and binds to repressive histone marks. In Chapter III, I show that ORCA binds to multiple repressive histone methyl transferases including G9a, GLP, Suv39h1 (H3K9me2/3), Suv420h1/h2 (H4K20me2/3) and EZH2 (H3K27me3). Removal of ORCA from human cells causes aberrations in the chromatin architecture. I therefore propose that ORCA acts as a scaffold protein that enables the formation of multiple histone lysine methyltransferase complexes at heterochromatic sites thereby facilitating chromatin organization.
Open chromatin structures regulate the efficiency of preRC formation and replication initiation. However, the molecular mechanisms that affect chromatin structure and how the preRC components establish…
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Prasanth%2C%20Supriya%20%20G%22%29&pagesize-30">Prasanth, Supriya G (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Prasanth%2C%20Supriya%20%20G%22%29&pagesize-30">Prasanth, Supriya G (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Stubbs%2C%20Lisa%20J%22%29&pagesize-30">Stubbs, Lisa J (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Belmont%2C%20Andrew%20%20S%22%29&pagesize-30">Belmont, Andrew S (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Freeman%2C%20Brian%20C%22%29&pagesize-30">Freeman, Brian C (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (committee member).
Subjects/Keywords: Origin Recognition Complex-Associated (ORCA); Origin recognition complex (ORC); DNA Replication; Chromatin
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APA ·
Chicago ·
MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Giri, S. (2016). Role of ORCA and ORC in chromatin organization and DNA replication. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/90477
Chicago Manual of Style (16th Edition):
Giri, Sumanprava. “Role of ORCA and ORC in chromatin organization and DNA replication.” 2016. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/90477.
MLA Handbook (7th Edition):
Giri, Sumanprava. “Role of ORCA and ORC in chromatin organization and DNA replication.” 2016. Web. 01 Mar 2021.
Vancouver:
Giri S. Role of ORCA and ORC in chromatin organization and DNA replication. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2016. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/90477.
Council of Science Editors:
Giri S. Role of ORCA and ORC in chromatin organization and DNA replication. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2016. Available from: http://hdl.handle.net/2142/90477
University of Illinois – Urbana-Champaign
30.
Ma, Wen.
Theoretical and computational investigations into structure-function relationships of biomolecular machines at atomic resolution.
Degree: PhD, Biophysics & Computnl Biology, 2017, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/98292
► Molecular machines play vital roles in many cellular processes, including DNA replication and repair, gene expression, protein degradation, protein secretion and maintenance of pH homeostasis.…
(more)
▼ Molecular machines play vital roles in many cellular processes, including DNA replication and repair, gene expression, protein degradation, protein secretion and maintenance of pH homeostasis. Despite intense research efforts, the atomic-level mechanism transmitting other form of energy into mechanical force is still unclear. Molecular dynamics simulations, though capable of providing atomic details, are limited in the study of molecular machines owing to the challenge that these machine usually function on a millisecond (or longer) time scale which, for a long time, could not be covered computationally. Employing advanced sampling techniques and theoretical modeling, we investigate the mechanism of three exemplary molecular machines in the following.
Transcription termination factor Rho is a key factor in bacterial gene expression and regulation. An essential question is how RNA is translocated by Rho using energy from ATP hydrolysis. By combining MD simulations with path sampling techniques and milestoning analysis, we find that the release of hydrolysis product (ADP+Pi) triggers the force-generating process of Rho through a 0.1 millisecond-long conformational transition. Our results not only reveal in new detail the mechanism employed by ring-shaped ATPase motors, for example the use of loosely bound and tightly bound hydrolysis reactant and product states to coordinate motor action, but also provide an effective approach to identify allosteric sites of multimeric enzymes in general.
Following the footstep of the study on Rho, we investigate a DNA helicase UvrD, which plays key roles in DNA replication and repair, by unwinding nucleic acid strands. Combining bioinformatics approaches and free energy calculations, we characterize how the UvrD helicase changes its conformation at the fork junction to switch its function from unwinding to rezipping DNA. The obtained transition pathway shows that UvrD opens the interface between the 2B/1B domains, allowing the bound ssDNA strand to escape and the other strand to bind to the ssDNA-binding domains. An interesting “tilted” conformation is revealed, which serves as a key metastable state for the ssDNA strand exchange. The simulation results not only match the single-molecule measurements from our collaborators, but also decipher key elements for the “hyper-helicase” behavior induced by a mutant (UvrD303).
The last project is a study of the growth of flagellum, which is an hours-long process. The building blocks of flagella, flagellin monomers, are pumped by a type III secretion system, through the flagellar interior channel to the growing tip. After a flagellin monomer binds to the tip of the filament, the growing flagellum is extended. The flagellin translocation process, due to the flagellum maximum length of 20 μm, is an extreme example of protein transport through channels. By deriving a theoretical model complemented by molecular dynamics simulations, we explain why the growth rate of flagellar filaments decays exponentially with filament length and why…
Advisors/Committee Members: Champaign%22%20%2Bcontributor%3A%28%22Chemla%2C%20Yann%20R%22%29&pagesize-30">Chemla, Yann R (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Schulten%2C%20Klaus%22%29&pagesize-30">Schulten, Klaus (advisor),
Champaign%22%20%2Bcontributor%3A%28%22Chemla%2C%20Yann%20R%22%29&pagesize-30">Chemla, Yann R (Committee Chair),
Champaign%22%20%2Bcontributor%3A%28%22Luthey-Schulten%2C%20Zaida%22%29&pagesize-30">Luthey-Schulten, Zaida (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Ha%2C%20Taekjip%22%29&pagesize-30">Ha, Taekjip (committee member),
Champaign%22%20%2Bcontributor%3A%28%22Tajkhorshid%2C%20Emad%22%29&pagesize-30">Tajkhorshid, Emad (committee member).
Subjects/Keywords: Molecular motor; Free energy landscape; Rate calculations; Transition pathway; Molecular dynamics; Multiscale modeling; Enhanced sampling methods; Helicase; ATPase
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Ma, W. (2017). Theoretical and computational investigations into structure-function relationships of biomolecular machines at atomic resolution. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/98292
Chicago Manual of Style (16th Edition):
Ma, Wen. “Theoretical and computational investigations into structure-function relationships of biomolecular machines at atomic resolution.” 2017. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed March 01, 2021.
http://hdl.handle.net/2142/98292.
MLA Handbook (7th Edition):
Ma, Wen. “Theoretical and computational investigations into structure-function relationships of biomolecular machines at atomic resolution.” 2017. Web. 01 Mar 2021.
Vancouver:
Ma W. Theoretical and computational investigations into structure-function relationships of biomolecular machines at atomic resolution. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2017. [cited 2021 Mar 01].
Available from: http://hdl.handle.net/2142/98292.
Council of Science Editors:
Ma W. Theoretical and computational investigations into structure-function relationships of biomolecular machines at atomic resolution. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2017. Available from: http://hdl.handle.net/2142/98292
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Open Access Theses and Dissertations