+publisher:"University of North Carolina" +contributor:("Pielak, Gary J.")

University of North Carolina

1. Barnes, Christopher O. Protein in-cell NMR in Escherichia coli.

Degree: Chemistry, 2010, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:86bacd50-a90b-43e9-aa46-0c286620f7b7

► The inside of the cell is a crowded and complex environment that is impossible to duplicate by studying proteins and other molecules in dilute solution.… (more)

Subjects/Keywords: College of Arts and Sciences; Department of Chemistry

Record Details Similar Records Cite « Share

❌

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Barnes, C. O. (2010). Protein in-cell NMR in Escherichia coli. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:86bacd50-a90b-43e9-aa46-0c286620f7b7

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Barnes, Christopher O. “Protein in-cell NMR in Escherichia coli.” 2010. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:86bacd50-a90b-43e9-aa46-0c286620f7b7.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Barnes, Christopher O. “Protein in-cell NMR in Escherichia coli.” 2010. Web. 18 Jan 2021.

Vancouver:

Barnes CO. Protein in-cell NMR in Escherichia coli. [Internet] [Thesis]. University of North Carolina; 2010. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:86bacd50-a90b-43e9-aa46-0c286620f7b7.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Barnes CO. Protein in-cell NMR in Escherichia coli. [Thesis]. University of North Carolina; 2010. Available from: https://cdr.lib.unc.edu/record/uuid:86bacd50-a90b-43e9-aa46-0c286620f7b7

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

2. Slade, Kristin M. Protein diffusion in Escherichia coli.

Degree: Chemistry, 2009, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:6861cac7-4086-4538-ae91-b093a6c7699d

► This dissertation describes the creation of a system to provide insight about the affects of protein expression on intracellular diffusion. Fluorescence recovery after photobleaching (FRAP)… (more)

▼ This dissertation describes the creation of a system to provide insight about the affects of protein expression on intracellular diffusion. Fluorescence recovery after photobleaching (FRAP) is used to obtain diffusion coefficients. Nuclear magnetic resonance spectroscopy detection of 19F isotopic-enriched proteins and fluorescent labeling with FlAsH and were attemped before settling on FRAP studies with green fluorecent protein (GFP). To regulate protein levels in Escherichia coli, several vector and promoter combinations were tested. Eventually, a single vector was created containing the structural gene for GFP under the lac promoter and a test protein under the araBAD promoter. With this vector, the test protein was expressed at varying levels and GFP was expressed at a constant level. Although chymotrypsin inhibitor 2 was originally chosen as the test protein, it was quickly replaced by alpha-synuclein, maltose binding protein, tau-40, and calmodulin. My most important result is that regardless of the type or amount of protein that was co-expressed, the GFP diffusion coefficient remained constant. We conclude that expression of these soluble proteins has little to no effect on the diffusion of GFP. Several disadvantages of FRAP became apparent in the process of obtaining these data. FRAP and the other common techniques for measuring translational diffusion, present numerous obstacles when working with structures that are only slightly larger than optical resolution, such as Escherichia coli cells. To overcome these obstacles, I also developed a new method to assess diffusion using through-prism total internal reflection fluorescence microscopy with continuous photobleaching. Here, the theoretical basis for this technique is presented. I demonstrate its applicability by measuring the diffusion coefficient, 6.3 ± 1.1 µm2/sec, of GFP in Escherichia coli cells. Advisors/Committee Members: Slade, Kristin M., Pielak, Gary J., University of North Carolina at Chapel Hill.

Subjects/Keywords: College of Arts and Sciences; Department of Chemistry

Record Details Similar Records Cite « Share

❌

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Slade, K. M. (2009). Protein diffusion in Escherichia coli. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:6861cac7-4086-4538-ae91-b093a6c7699d

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Slade, Kristin M. “Protein diffusion in Escherichia coli.” 2009. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:6861cac7-4086-4538-ae91-b093a6c7699d.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Slade, Kristin M. “Protein diffusion in Escherichia coli.” 2009. Web. 18 Jan 2021.

Vancouver:

Slade KM. Protein diffusion in Escherichia coli. [Internet] [Thesis]. University of North Carolina; 2009. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:6861cac7-4086-4538-ae91-b093a6c7699d.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Slade KM. Protein diffusion in Escherichia coli. [Thesis]. University of North Carolina; 2009. Available from: https://cdr.lib.unc.edu/record/uuid:6861cac7-4086-4538-ae91-b093a6c7699d

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

3. Ruf, Rebecca A. S. Tyrosine and the oxidative aggregation of alpha-synuclein.

Degree: Chemistry, 2009, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:d6690e11-2176-477c-954b-82292a4fbec2

► Oxidative stress and aggregation of the protein alpha-synuclein are thought to be key factors in Parkinson's disease. Previous work shows that cytochrome c plus H2O2… (more)

Subjects/Keywords: College of Arts and Sciences; Department of Chemistry

Record Details Similar Records Cite « Share

❌

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Ruf, R. A. S. (2009). Tyrosine and the oxidative aggregation of alpha-synuclein. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:d6690e11-2176-477c-954b-82292a4fbec2

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Ruf, Rebecca A S. “Tyrosine and the oxidative aggregation of alpha-synuclein.” 2009. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:d6690e11-2176-477c-954b-82292a4fbec2.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Ruf, Rebecca A S. “Tyrosine and the oxidative aggregation of alpha-synuclein.” 2009. Web. 18 Jan 2021.

Vancouver:

Ruf RAS. Tyrosine and the oxidative aggregation of alpha-synuclein. [Internet] [Thesis]. University of North Carolina; 2009. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:d6690e11-2176-477c-954b-82292a4fbec2.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Ruf RAS. Tyrosine and the oxidative aggregation of alpha-synuclein. [Thesis]. University of North Carolina; 2009. Available from: https://cdr.lib.unc.edu/record/uuid:d6690e11-2176-477c-954b-82292a4fbec2

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

4. Charlton, Lisa M. Protein behavior in crowded environments.

Degree: Chemistry, 2008, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:f717815f-a4bb-4f1d-9ff9-432e81e9032a

► The cell’s interior is a complex milieu where proteins exist in an environment crowded with other macromolecules. The influence of macromolecular crowding is prevalent in… (more)

Subjects/Keywords: College of Arts and Sciences; Department of Chemistry

Record Details Similar Records Cite « Share

❌

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Charlton, L. M. (2008). Protein behavior in crowded environments. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:f717815f-a4bb-4f1d-9ff9-432e81e9032a

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Charlton, Lisa M. “Protein behavior in crowded environments.” 2008. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:f717815f-a4bb-4f1d-9ff9-432e81e9032a.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Charlton, Lisa M. “Protein behavior in crowded environments.” 2008. Web. 18 Jan 2021.

Vancouver:

Charlton LM. Protein behavior in crowded environments. [Internet] [Thesis]. University of North Carolina; 2008. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:f717815f-a4bb-4f1d-9ff9-432e81e9032a.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Charlton LM. Protein behavior in crowded environments. [Thesis]. University of North Carolina; 2008. Available from: https://cdr.lib.unc.edu/record/uuid:f717815f-a4bb-4f1d-9ff9-432e81e9032a

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

5. Miklos, Andrew C. Macromolecular crowding effects on globular protein stability.

Degree: Chemistry, 2011, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:e423c83d-711b-4082-92e7-db06d0be4264

► Macromolecular crowding inside cells is proposed to change many aspects of proteins compared to dilute solution. As such, it is an increasingly studied topic, both… (more)

Subjects/Keywords: College of Arts and Sciences; Department of Chemistry

Record Details Similar Records Cite « Share

❌

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Miklos, A. C. (2011). Macromolecular crowding effects on globular protein stability. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:e423c83d-711b-4082-92e7-db06d0be4264

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Miklos, Andrew C. “Macromolecular crowding effects on globular protein stability.” 2011. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:e423c83d-711b-4082-92e7-db06d0be4264.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Miklos, Andrew C. “Macromolecular crowding effects on globular protein stability.” 2011. Web. 18 Jan 2021.

Vancouver:

Miklos AC. Macromolecular crowding effects on globular protein stability. [Internet] [Thesis]. University of North Carolina; 2011. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:e423c83d-711b-4082-92e7-db06d0be4264.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Miklos AC. Macromolecular crowding effects on globular protein stability. [Thesis]. University of North Carolina; 2011. Available from: https://cdr.lib.unc.edu/record/uuid:e423c83d-711b-4082-92e7-db06d0be4264

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

6. Schlesinger, Alexander P. Protein NMR in insect and bacterial cells.

Degree: Biochemistry and Biophysics, 2011, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:8780f339-1049-441c-bd14-ddf14c42dc99

► Proteins perform their functions in cells where macromolecular solutes can reach concentrations of >300 g/L and occupy >30% of the volume. The volume excluded by… (more)

▼ Proteins perform their functions in cells where macromolecular solutes can reach concentrations of >300 g/L and occupy >30% of the volume. The volume excluded by these macromolecules will stabilize globular proteins because the native state of a globular protein occupies less space than the denatured state. The idea that volume exclusion should fold globular proteins is tested in Escherichia coli by using protein L Kx7E, a 7-kDa unstable globular protein variant of wild-type protein L. This variant requires high concentrations of monovalent salts to fold. The standard unfolding free energy of Kx7E in the absence of NaCl is -1.0 kcal/mol as determined by using in vitro 1H-15N nuclear magnetic resonance spectroscopy. In-cell nuclear magnetic resonance spectroscopy with 19F-labeled Kx7E is used to show that the crowded cytoplasm of E. coli cannot overcome even this modest free energy deficit and fold the variant. The data are consistent with the idea that non-specific interactions between cytoplasmic components can overcome the excluded volume effect. Evidence for these interactions is provided by the observation that adding simple salts folds Kx7E in dilute solution, but increasing the salt concentration inside E. coli does not fold the variant. An attempt was made to acquire nuclear magnetic resonance spectra of the intrinsically disordered protein α-synuclein and the green fluorescent protein inside insect cells. Infection of Spodoptera frugiperda insect cells with recombinant baculoviruses containing a late basic protein promoter produces sufficient recombinant protein while maintaining cell viability for spectral acquisition. However, incorporation of the nuclear magnetic resonance active isotopes 15N or 19F into the recombinant proteins through the addition of 15NH4Cl or 5-fluorotryptophan to the cell culture media proved unsuccessful. The similarity of the spectra for α-synuclein, green fluorescent protein, and a fusion protein suggests isotope incorporation into intracellular metabolites. Thus, protein nuclear magnetic resonance spectra cannot be acquired in insect cells by using these experimental conditions. Advisors/Committee Members: Schlesinger, Alexander P., Pielak, Gary J., University of North Carolina at Chapel Hill.

Subjects/Keywords: School of Medicine; Department of Biochemistry and Biophysics

Record Details Similar Records Cite « Share

❌

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Schlesinger, A. P. (2011). Protein NMR in insect and bacterial cells. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:8780f339-1049-441c-bd14-ddf14c42dc99

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Schlesinger, Alexander P. “Protein NMR in insect and bacterial cells.” 2011. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:8780f339-1049-441c-bd14-ddf14c42dc99.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Schlesinger, Alexander P. “Protein NMR in insect and bacterial cells.” 2011. Web. 18 Jan 2021.

Vancouver:

Schlesinger AP. Protein NMR in insect and bacterial cells. [Internet] [Thesis]. University of North Carolina; 2011. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:8780f339-1049-441c-bd14-ddf14c42dc99.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Schlesinger AP. Protein NMR in insect and bacterial cells. [Thesis]. University of North Carolina; 2011. Available from: https://cdr.lib.unc.edu/record/uuid:8780f339-1049-441c-bd14-ddf14c42dc99

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

7. Wang, Yaqiang. Macromolecular crowding and protein chemistry: views from inside and outside cells.

Degree: Chemistry, 2012, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:d9c6b6bc-4e68-4734-986c-9900013622e5

► The cytoplasm is crowded, and the concentration of macromolecules can reach ~ 300 g/L, an environment vastly different from the dilute, idealized conditions usually used… (more)

▼ The cytoplasm is crowded, and the concentration of macromolecules can reach ~ 300 g/L, an environment vastly different from the dilute, idealized conditions usually used in biophysical studies. Macromolecular crowding arise from two phenomena, excluded volume and nonspecific chemical interactions, until recently, only excluded volume effect has been considered. Theory predicts that this macromolecular crowding can have large effects. Most proteins, however, are studied outside cells in dilute solution with macromolecule concentrations of 10 g/L or less. In-cell NMR provides a means to assess protein biophysics at atomic resolution in living cells, but it remains in its infancy, and several potential challenges need to be addressed. One challenge is the inability to observe 15N-1H NMR spectra from many small globular proteins. 19F NMR was used to expand the application of in-cell NMR. This work suggests that high viscosity and weak interactions in the cytoplasm can make routine 15N enrichment a poor choice for in-cell NMR studies of globular proteins in Escherichia coli. To gain insight into this problem, I turned to in vitro experiments where conditions can be controlled with precision. Using both synthetic polymers and globular proteins, I studied the effects of crowding on the diffusion of the test protein, chymotrypsin inhibitor 2. The results not only pinpoint the source of the problem - nonspecific chemical interactions - but also suggest that proteins are more suitable mimics of the intracellular environment. I also measured the stability of ubiquitin in solutions crowded with synthetic polymers or globular proteins to further elucidate the role of nonspecific chemical interactions under crowded conditions. The increased stability observed in synthetic crowders was consistent with a dominant entropic role for excluded volume, but the effect of protein crowders depended on charge. Protein-induced crowding increased stability when the sign of the net charge of the crowder was the same as that of ubiquitin, but decreased stability when the proteins were oppositely charged. The results indicate that synthetic polymers do not provide physiologically relevant insights and that the overall effect of macromolecular crowding depends on the winner of the near stalemate between excluded volume and nonspecific interactions. Advisors/Committee Members: Wang, Yaqiang, Pielak, Gary J..

Subjects/Keywords: College of Arts and Sciences; Department of Chemistry

Record Details Similar Records Cite « Share

❌

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Wang, Y. (2012). Macromolecular crowding and protein chemistry: views from inside and outside cells. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:d9c6b6bc-4e68-4734-986c-9900013622e5

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Wang, Yaqiang. “Macromolecular crowding and protein chemistry: views from inside and outside cells.” 2012. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:d9c6b6bc-4e68-4734-986c-9900013622e5.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Wang, Yaqiang. “Macromolecular crowding and protein chemistry: views from inside and outside cells.” 2012. Web. 18 Jan 2021.

Vancouver:

Wang Y. Macromolecular crowding and protein chemistry: views from inside and outside cells. [Internet] [Thesis]. University of North Carolina; 2012. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:d9c6b6bc-4e68-4734-986c-9900013622e5.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Wang Y. Macromolecular crowding and protein chemistry: views from inside and outside cells. [Thesis]. University of North Carolina; 2012. Available from: https://cdr.lib.unc.edu/record/uuid:d9c6b6bc-4e68-4734-986c-9900013622e5

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

8. Busan, Steven. Frameworks for large-scale RNA structure profiling in transcriptomes and disease.

Degree: Chemistry, 2015, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:fa2d2fe1-0129-4227-a872-ce956896a54e

► In addition to their role as intermediaries on the route to protein synthesis, RNA molecules have long been known to base-pair into complex structures that… (more)

Subjects/Keywords: Chemistry; College of Arts and Sciences; Department of Chemistry

Record Details Similar Records Cite « Share

❌

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Busan, S. (2015). Frameworks for large-scale RNA structure profiling in transcriptomes and disease. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:fa2d2fe1-0129-4227-a872-ce956896a54e

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Busan, Steven. “Frameworks for large-scale RNA structure profiling in transcriptomes and disease.” 2015. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:fa2d2fe1-0129-4227-a872-ce956896a54e.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Busan, Steven. “Frameworks for large-scale RNA structure profiling in transcriptomes and disease.” 2015. Web. 18 Jan 2021.

Vancouver:

Busan S. Frameworks for large-scale RNA structure profiling in transcriptomes and disease. [Internet] [Thesis]. University of North Carolina; 2015. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:fa2d2fe1-0129-4227-a872-ce956896a54e.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Busan S. Frameworks for large-scale RNA structure profiling in transcriptomes and disease. [Thesis]. University of North Carolina; 2015. Available from: https://cdr.lib.unc.edu/record/uuid:fa2d2fe1-0129-4227-a872-ce956896a54e

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

9. Rice, Greggory. High-throughput Experiment Driven Modeling of RNA Interactions and Structures.

Degree: Chemistry, 2015, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:cbeb11b0-d2f3-4f68-8945-5a924e10bd59

► The higher order structure of an RNA is often essential to its biological function, modulating its interactions with ligands, protein partners, and other RNAs. Modeling… (more)

▼ The higher order structure of an RNA is often essential to its biological function, modulating its interactions with ligands, protein partners, and other RNAs. Modeling RNA secondary structure, assessing the accuracy of RNA structural models, and discovering new functional motifs are challenging problems that are confounded by the length and complexity of the studied RNA. Improvements in structure modeling accuracy can be made by incorporating SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) and DMS (dimethyl sulfate) chemical probing data, however these models remain imperfect. In this work I apply principals of molecular modeling in interpret chemical probing experiments and create analytical and experimental tools that enable large-scale experiment-driven modeling of RNA interactions and structures. First, I use electronic structure modeling to propose a mechanism to explain the preferential reactivity of the SHAPE reagent 1M6 at nucleotides exhibiting an available open stack in folded RNAs. I also use molecular modeling at the nucleotide level to develop a model that accurately predicts the disruptive effects of SHAPE adducts on RNA tertiary structure. Second, I create a new energy potential for RNA structure prediction using information from a three-reagent SHAPE experiment that increases the accuracy of modeling accuracy from 85% to above 90% for some of the most difficult-to-predict RNA structures. Third, working collaboratively with others in the lab, I validate a new approach for melding SHAPE chemical probing with deep sequencing in a new technique termed SHAPE mutational profiling (SHAPE-MaP). The ability to quickly generate structural data for RNAs of unprecedented size using SHAPE-MaP presents a new challenge: accurately modeling large RNA secondary structures. To solve this problem I develop software, called SuperFold, which uses a windowed modeling algorithm to enable rapid secondary structure prediction and discovery of the most stable structural motifs in long RNAs. Fourth, I extend the RING-MaP experiment and analysis to enable use with random primers and applied it to the bacterial ribosome. With the improved RING-MaP experiment I am able to detect structural interaction networks within the small ribosomal subunit. Additionally, I am able to perturb those structural networks by adding the antibiotic spectinomycin. Coupled together, the work presented here will provide valuable tools that democratize RNA structure analysis and help others in the RNA community understand the role of RNA structure at new and exciting scales. Advisors/Committee Members: Rice, Greggory, Weeks, Kevin, Pielak, Gary J., Laederach, Alain, Dokholyan, Nikolay, Lawrence, David.

Subjects/Keywords: Biochemistry; Bioinformatics; Chemistry; College of Arts and Sciences; Department of Chemistry

Record Details Similar Records Cite « Share

❌

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Rice, G. (2015). High-throughput Experiment Driven Modeling of RNA Interactions and Structures. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:cbeb11b0-d2f3-4f68-8945-5a924e10bd59

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Rice, Greggory. “High-throughput Experiment Driven Modeling of RNA Interactions and Structures.” 2015. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:cbeb11b0-d2f3-4f68-8945-5a924e10bd59.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Rice, Greggory. “High-throughput Experiment Driven Modeling of RNA Interactions and Structures.” 2015. Web. 18 Jan 2021.

Vancouver:

Rice G. High-throughput Experiment Driven Modeling of RNA Interactions and Structures. [Internet] [Thesis]. University of North Carolina; 2015. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:cbeb11b0-d2f3-4f68-8945-5a924e10bd59.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Rice G. High-throughput Experiment Driven Modeling of RNA Interactions and Structures. [Thesis]. University of North Carolina; 2015. Available from: https://cdr.lib.unc.edu/record/uuid:cbeb11b0-d2f3-4f68-8945-5a924e10bd59

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

10. Sarkar, Mohona. Effects of Biomolecular Crowding on Protein Stability.

Degree: Chemistry, 2014, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:02986878-4211-42ea-8f4b-c1fb45238da4

► The intracellular milieu is complex, heterogeneous and crowded – an environment vastly different from dilute, buffered solutions where most biophysical studies are performed. The cytoplasm excludes… (more)

▼ The intracellular milieu is complex, heterogeneous and crowded – an environment vastly different from dilute, buffered solutions where most biophysical studies are performed. The cytoplasm excludes about a third of the volume available to macromolecules in dilute solution. This exclusion arises from the sum of two phenomena: steric repulsions and chemical interactions, often called hard and soft interactions, respectively. Most efforts to understand crowding have focused on steric repulsions. Globular protein stability is the difference in free energy between the compact, biologically functional native state and the ensemble of less compact, nonfunctional denatured state. The hard-core repulsive component of crowding stabilizes globular proteins because the decrease in available volume favors compact species. The effect of soft interactions can be stabilizing or destabilizing. Soft repulsive interactions reinforce the stabilizing influence of hard-core repulsions. However, the equilibrium is shifted towards the denatured state in systems dominated by attractive non-specific interactions, because unfolding exposes more reactive surface. In Chapter 1, I introduce the concepts of hard and soft interactions in more depth and discuss how they are expected to affect the equilibrium thermodynamic stability of globular proteins. In Chapter 2, I describe experiments that test these concepts by using Escherichia coli cell lysates as the crowding agents, chymotrypsin inhibitor 2 (CI2) as the test protein and NMR-detected amide-proton exchange to measure stability. The lysate destabilizes CI2, and the destabilization increases with increasing lysate concentration. This observation shows that the cytoplasm interacts favorably, but non-specifically, with CI2, and these interactions overcome the stabilizing hard-core repulsions. In fact, the effects of the lysate are even stronger than those of homogeneous protein crowders, reinforcing the biological significance of weak, non-specific interactions. In Chapter 3, I test the idea that the net charge on the crowding proteins affects stability. To accomplish this goal, I isolated the anionic proteins from the lysate and used them as the crowding agent. CI2 is an anion under the chosen conditions, and, therefore, I expected the net repulsive interactions between CI2 and the crowders to increase the stability of CI2. Instead, the refined lysate also resulted in destabilization. Thus, even the anionic proteins, which have the same net charge as CI2, significantly interact with CI2's backbone non-specifically to overcome the stabilizing effect of steric repulsion. My in vitro studies show that weak chemical interactions play key roles in the cytosol. It will even be more difficult to identify soft interactions in living cells where reductionist approaches are difficult or impossible to apply. Nevertheless, endeavors aimed at quantifying soft interactions are essential for producing a physiologically relevant understanding of biophysics. Once the details of soft interactions are known, it… Advisors/Committee Members: Sarkar, Mohona, Pielak, Gary J..

Subjects/Keywords: College of Arts and Sciences; Department of Chemistry

Record Details Similar Records Cite « Share

❌

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Sarkar, M. (2014). Effects of Biomolecular Crowding on Protein Stability. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:02986878-4211-42ea-8f4b-c1fb45238da4

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Sarkar, Mohona. “Effects of Biomolecular Crowding on Protein Stability.” 2014. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:02986878-4211-42ea-8f4b-c1fb45238da4.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Sarkar, Mohona. “Effects of Biomolecular Crowding on Protein Stability.” 2014. Web. 18 Jan 2021.

Vancouver:

Sarkar M. Effects of Biomolecular Crowding on Protein Stability. [Internet] [Thesis]. University of North Carolina; 2014. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:02986878-4211-42ea-8f4b-c1fb45238da4.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Sarkar M. Effects of Biomolecular Crowding on Protein Stability. [Thesis]. University of North Carolina; 2014. Available from: https://cdr.lib.unc.edu/record/uuid:02986878-4211-42ea-8f4b-c1fb45238da4

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

11. Smith, Austin. PROTEIN STRUCTURE, STABILITY AND DYNAMICS IN CELLS AND CELL-LIKE ENVIRONMENTS.

Degree: Chemistry, 2015, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:19a082d1-c0ab-4254-a131-5445a0502aed

► The intracellular milieu is filled with small molecules, nucleic acids, lipids and proteins. Theories have attempted to explain how macromolecules react to this environment for… (more)

Subjects/Keywords: Chemistry; Biophysics; Biochemistry; College of Arts and Sciences; Department of Chemistry

Record Details Similar Records Cite « Share

❌

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Smith, A. (2015). PROTEIN STRUCTURE, STABILITY AND DYNAMICS IN CELLS AND CELL-LIKE ENVIRONMENTS. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:19a082d1-c0ab-4254-a131-5445a0502aed

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Smith, Austin. “PROTEIN STRUCTURE, STABILITY AND DYNAMICS IN CELLS AND CELL-LIKE ENVIRONMENTS.” 2015. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:19a082d1-c0ab-4254-a131-5445a0502aed.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Smith, Austin. “PROTEIN STRUCTURE, STABILITY AND DYNAMICS IN CELLS AND CELL-LIKE ENVIRONMENTS.” 2015. Web. 18 Jan 2021.

Vancouver:

Smith A. PROTEIN STRUCTURE, STABILITY AND DYNAMICS IN CELLS AND CELL-LIKE ENVIRONMENTS. [Internet] [Thesis]. University of North Carolina; 2015. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:19a082d1-c0ab-4254-a131-5445a0502aed.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Smith A. PROTEIN STRUCTURE, STABILITY AND DYNAMICS IN CELLS AND CELL-LIKE ENVIRONMENTS. [Thesis]. University of North Carolina; 2015. Available from: https://cdr.lib.unc.edu/record/uuid:19a082d1-c0ab-4254-a131-5445a0502aed

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

12. Treadway, James. Synthesis and Chromatographic Evaluation of Superficially Porous Particles for Ultrahigh Pressure Liquid Chromatography.

Degree: Chemistry, 2015, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:f7bdff89-494d-4a01-8c40-ccac5f087af5

► Chromatographic separations play a vital role in the separation and analysis of biological samples. Superficially porous particle have emerged as a useful particle architecture for… (more)

▼ Chromatographic separations play a vital role in the separation and analysis of biological samples. Superficially porous particle have emerged as a useful particle architecture for liquid chromatography. These particles have received significant attention recently for the chromatographic efficiency gains that they display. Ultrahigh pressure liquid chromatography employs small particles and high pressures to also yield highly efficient separations. Increasingly, the field of chromatography had employed a combination of these two methods for creating highly efficient columns. One portion of this work details the work with micron-sized superficially porous particles for the separation of intact proteins and other large molecules. Superficially porous particles specifically tailored for the separation of large molecules are not currently commercially available in the single micron size range. One aspect of this work describes the synthetic route that was utilized to create these particles in such a small overall particle size. Another aspect of this work details an evaluation of the separation capabilities of these particles when packed into capillary columns. These columns were utilized for the separation of small molecules, peptides and proteins. Another portion of this work concerns the utilization of commercially manufactured superficially porous particles. One chapter focuses on the creation of highly efficient capillary columns packed with commercial prototype superficially porous particles. The capillary columns created in that chapter exhibit isocratic efficiencies for the separation of small molecules rivaling those seen for capillary columns packed with fully porous particles. This is the first time that such efficient separations have been demonstrated using superficially porous particles packed into capillary columns. A final section of the dissertation focuses on the comparison between commercially available fully and superficially porous particles packed into capillary columns. These particles were bonded with either conventional or charged-surface reversed-phase bondings. The separation characteristics for these columns were evaluated in terms of separating small molecules, peptides, and proteins. It was seen that both the bonded phase and the particle morphology contributed to separation differences between the columns. This work provides insight into which particle and bonding type should be pursued for future investigations. Advisors/Committee Members: Treadway, James, Jorgenson, James, Schoenfisch, Mark H., Ramsey, J. Michael, Pielak, Gary J., Erie, Dorothy.

Subjects/Keywords: Chemistry, Analytic; Chemistry; College of Arts and Sciences; Department of Chemistry

Record Details Similar Records Cite « Share

❌

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Treadway, J. (2015). Synthesis and Chromatographic Evaluation of Superficially Porous Particles for Ultrahigh Pressure Liquid Chromatography. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:f7bdff89-494d-4a01-8c40-ccac5f087af5

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Treadway, James. “Synthesis and Chromatographic Evaluation of Superficially Porous Particles for Ultrahigh Pressure Liquid Chromatography.” 2015. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:f7bdff89-494d-4a01-8c40-ccac5f087af5.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Treadway, James. “Synthesis and Chromatographic Evaluation of Superficially Porous Particles for Ultrahigh Pressure Liquid Chromatography.” 2015. Web. 18 Jan 2021.

Vancouver:

Treadway J. Synthesis and Chromatographic Evaluation of Superficially Porous Particles for Ultrahigh Pressure Liquid Chromatography. [Internet] [Thesis]. University of North Carolina; 2015. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:f7bdff89-494d-4a01-8c40-ccac5f087af5.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Treadway J. Synthesis and Chromatographic Evaluation of Superficially Porous Particles for Ultrahigh Pressure Liquid Chromatography. [Thesis]. University of North Carolina; 2015. Available from: https://cdr.lib.unc.edu/record/uuid:f7bdff89-494d-4a01-8c40-ccac5f087af5

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

13. Lujan, Scott. Bacterial Conjugation and its Inhibition: The Hows and Whys of Conjugation and What Can be Done to Control It.

Degree: Biochemistry and Biophysics, 2008, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:479d9a87-b137-4371-b5de-c871ab80e258

► Conjugation is the primary vehicle for the horizontal transfer virulence factor genes, such as antibiotic resistance, within and between bacterial strains. In certain epicenters, such… (more)

▼ Conjugation is the primary vehicle for the horizontal transfer virulence factor genes, such as antibiotic resistance, within and between bacterial strains. In certain epicenters, such as hospitals in less developed parts of the world, immune-compromised patients and misuse of antibiotics combine to select for the development and dissemination of these pathogenicity factors via conjugation. Inhibition of conjugation would prove a boon for curbing the creation and spread of new virulent or multi-drug resistant strains. DNA relaxases are the keystone proteins of each conjugative system. TraI is the relaxase of the F plasmid, the archetypal model system for conjugation. Toward revelation and inhibition of relaxase mechanisms, I used bioinformatics and limited proteolysis to find and identify new domains on the F TraI enzyme. I then solved TraI/DNA co-crystal structures that showed a novel DNA binding mode. Based on structure comparisons, sequence conservation, and mutant activity studies, I proposed a mechanism for TraI activity that required the existence of a dual phosphotyrosine intermediate, which has since been observed. I then demonstrated that bisphosphonate compounds mimicking this intermediate are nanomolar in vitro TraI inhibitors. I determined the relaxase structure in complex with one such inhibitor, bound as predicted. We showed that several of these compounds are potent in cell inhibitors of conjugation that often selectively kill conjugation-capable cells, a novel antibiotic paradigm. We showed that oral treatment of gnotobiotic mice with two inhibitors, clodronate and etidronate, decreased the gastrointestinal F+ bacterial load twentyfold without apparent side effects. Beyond the model system, etidronate showed selective in cell potency versus cells harboring a known clinical resistance-bearing R100 plasmid. Etidronate and clodronate are already clinically approved for the treatment of bone loss in humans. Toward more general relaxase inhibition phylogenetic analyses and studies with other medically relevant relaxases, including viral replicative relaxases, are ongoing. Advisors/Committee Members: Lujan, Scott, Redinbo, Matthew R., Wolfenden, Richard, Pielak, Gary J., Matson, Steven, Temple, Brenda.

Subjects/Keywords: School of Medicine; Department of Biochemistry and Biophysics

Record Details Similar Records Cite « Share

❌

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Lujan, S. (2008). Bacterial Conjugation and its Inhibition: The Hows and Whys of Conjugation and What Can be Done to Control It. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:479d9a87-b137-4371-b5de-c871ab80e258

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Lujan, Scott. “Bacterial Conjugation and its Inhibition: The Hows and Whys of Conjugation and What Can be Done to Control It.” 2008. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:479d9a87-b137-4371-b5de-c871ab80e258.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Lujan, Scott. “Bacterial Conjugation and its Inhibition: The Hows and Whys of Conjugation and What Can be Done to Control It.” 2008. Web. 18 Jan 2021.

Vancouver:

Lujan S. Bacterial Conjugation and its Inhibition: The Hows and Whys of Conjugation and What Can be Done to Control It. [Internet] [Thesis]. University of North Carolina; 2008. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:479d9a87-b137-4371-b5de-c871ab80e258.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Lujan S. Bacterial Conjugation and its Inhibition: The Hows and Whys of Conjugation and What Can be Done to Control It. [Thesis]. University of North Carolina; 2008. Available from: https://cdr.lib.unc.edu/record/uuid:479d9a87-b137-4371-b5de-c871ab80e258

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

14. Clarkson, Michael. Dynamic Effects of Mutations in Eglin c.

Degree: Biochemistry and Biophysics, 2006, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:5a070fe7-5aff-4908-9e6c-782a95fe1a25

► In order to investigate the mechanism of dynamic propagation in proteins and the role these responses play in energetic communication, a series of novel dynamic… (more)

Subjects/Keywords: School of Medicine; Department of Biochemistry and Biophysics

Record Details Similar Records Cite « Share

❌

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Clarkson, M. (2006). Dynamic Effects of Mutations in Eglin c. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:5a070fe7-5aff-4908-9e6c-782a95fe1a25

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Clarkson, Michael. “Dynamic Effects of Mutations in Eglin c.” 2006. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:5a070fe7-5aff-4908-9e6c-782a95fe1a25.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Clarkson, Michael. “Dynamic Effects of Mutations in Eglin c.” 2006. Web. 18 Jan 2021.

Vancouver:

Clarkson M. Dynamic Effects of Mutations in Eglin c. [Internet] [Thesis]. University of North Carolina; 2006. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:5a070fe7-5aff-4908-9e6c-782a95fe1a25.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Clarkson M. Dynamic Effects of Mutations in Eglin c. [Thesis]. University of North Carolina; 2006. Available from: https://cdr.lib.unc.edu/record/uuid:5a070fe7-5aff-4908-9e6c-782a95fe1a25

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

15. Cohen, Rachel. Quinary structure alters protein folding landscapes.

Degree: Chemistry, 2017, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:7b21bc35-13d7-417c-856d-683de8dd6205

► Most knowledge of protein chemistry is derived from experiments performed in dilute, buffered solutions. Although such experiments provide essential information, proteins function in the crowded… (more)

▼ Most knowledge of protein chemistry is derived from experiments performed in dilute, buffered solutions. Although such experiments provide essential information, proteins function in the crowded and complex cellular environment, which imposes an additional level of structure. This quinary structure comprises the transient interactions between macromolecules that provides organization and compartmentalization inside cells. I have used in-cell NMR spectroscopy to characterize quinary structure, and have shown that globular protein stability is affected by quinary interactions involving both the folded state and the unfolded ensemble. Chapter 1 reviews the history of quinary structure in the context of metabolic pathways, and the technological advances that have yielded recent insight into protein behavior in living cells. In Chapter 2, I use the K10H variant of the B domain of protein G (GB1, 6.2 kDa) as a pH reporter in Escherichia coli cells to show that quinary interactions influence the quality of in-cell 15N–1H HSQC NMR spectra. In Chapter 3, I quantify the pH-dependence of GB1 stability in cells. At neutral pH, GB1 stability in cells is comparable to that in buffer. As the pH decreases, the increased number of attractive interactions between E. coli proteins and GB1 destabilizes GB1 relative to buffer alone. I conclude that electrostatic interactions involving surface residues of the folded state contribute to quinary structure. Chapter 4 shows that quinary structure can also affect the unfolded state ensemble. It has been known for many years that the unfolded ensemble of GB1 is stabilized by a non-native hydrophobic staple. Exploiting this idea, I made several mutations that do not change the folded state of GB1, but have a large effect on its stability in buffer. These effects are severely attenuated in cells, demonstrating that the cellular environment can remodel the unfolded ensemble. My work shows that there is more to protein stability than a well-packed hydrophobic core; the key to understanding protein behavior in nature lies in quinary structure. Advisors/Committee Members: Cohen, Rachel, Pielak, Gary J., Brustad, Eric, Redinbo, Matthew R., Riordan, John, Lee, Andrew.

Subjects/Keywords: College of Arts and Sciences; Department of Chemistry

Record Details Similar Records Cite « Share

❌

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Cohen, R. (2017). Quinary structure alters protein folding landscapes. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:7b21bc35-13d7-417c-856d-683de8dd6205

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Cohen, Rachel. “Quinary structure alters protein folding landscapes.” 2017. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:7b21bc35-13d7-417c-856d-683de8dd6205.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Cohen, Rachel. “Quinary structure alters protein folding landscapes.” 2017. Web. 18 Jan 2021.

Vancouver:

Cohen R. Quinary structure alters protein folding landscapes. [Internet] [Thesis]. University of North Carolina; 2017. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:7b21bc35-13d7-417c-856d-683de8dd6205.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Cohen R. Quinary structure alters protein folding landscapes. [Thesis]. University of North Carolina; 2017. Available from: https://cdr.lib.unc.edu/record/uuid:7b21bc35-13d7-417c-856d-683de8dd6205

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

16. Falk, Bradley. Probing subunit communication via a singly ligated state of dimeric thymidylate synthase.

Degree: Biochemistry and Biophysics, 2017, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:5277b259-725a-470f-a204-5de0c349ead6

► Enzymes are highly dynamic molecules that undergo motions on a wide variety of timescales that are linked to all aspects of their function from mediating… (more)

▼ Enzymes are highly dynamic molecules that undergo motions on a wide variety of timescales that are linked to all aspects of their function from mediating substrate binding and enabling chemistry to facilitating regulation by effector ligands. NMR studies on a handful of small monomeric enzymes have highlighted the role of dynamics in simple systems, but most enzymes possess added complexity stemming from size, oligomeric structures, multistep and/or multisubstrate mechanisms, and allosteric regulation. In recent years there has been a resurgence of interest in understanding the mechanisms of protein allostery. Canonically, allostery occurs in oligomeric proteins: ligand binding in one subunit alters binding or activity in a symmetry related second subunit. For such systems it is often straightforward to measure cooperative binding thermodynamics, but gaining detailed structural and/or dynamic mechanisms for intersubunit communication has proved to be much more difficult. This work has focused on applying NMR techniques to probe the inner workings of cooperativity and intersubunit communication in a large dimeric enzyme, Escherichia coli thymidylate synthase. A combined ITC and NMR approach showed that binding of substrate dUMP and cofactor mTHF occurs in both binding sites and dUMP binds with equal affinity. However, the two dUMP binding events are characterized by slightly different ΔCp showing that the two binding sites are not formally equivalent and presenting evidence for intersubunit communication. To probe this inequivalence a pair of mixed labeled dimers were created that can only bind substrate in one subunit and can be labeled on either subunit for NMR studies. These mixed labeled dimers enabled an NMR approach to distinguish the step-wise effects of ligand binding to this dimeric enzyme with subunit specificity. Binding studies revealed an asymmetric response to the two dUMP binding events and evidence of an extreme state in the singly bound form. The mixed dimers allow for visualization of singly bound peak positions which are typically elusive for dimeric systems. In thymidylate synthase these singly bound peaks fall in unexpected patterns that yield new insight into intersubunit communication. Advisors/Committee Members: Falk, Bradley, Lee, Andrew, Carter, Charles, Pielak, Gary J., Traut, Thomas, Zhang, Qi.

Subjects/Keywords: School of Medicine; Department of Biochemistry and Biophysics

Record Details Similar Records Cite « Share

❌

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Falk, B. (2017). Probing subunit communication via a singly ligated state of dimeric thymidylate synthase. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:5277b259-725a-470f-a204-5de0c349ead6

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Falk, Bradley. “Probing subunit communication via a singly ligated state of dimeric thymidylate synthase.” 2017. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:5277b259-725a-470f-a204-5de0c349ead6.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Falk, Bradley. “Probing subunit communication via a singly ligated state of dimeric thymidylate synthase.” 2017. Web. 18 Jan 2021.

Vancouver:

Falk B. Probing subunit communication via a singly ligated state of dimeric thymidylate synthase. [Internet] [Thesis]. University of North Carolina; 2017. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:5277b259-725a-470f-a204-5de0c349ead6.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Falk B. Probing subunit communication via a singly ligated state of dimeric thymidylate synthase. [Thesis]. University of North Carolina; 2017. Available from: https://cdr.lib.unc.edu/record/uuid:5277b259-725a-470f-a204-5de0c349ead6

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

17. Monteith, William. Residue Level Quantification of Protein Stability in Living Cells.

Degree: Chemistry, 2014, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:2015b666-3d6f-4fce-98c8-d7f182ea4426

► The intracellular milieu differs from the dilute conditions in which most biophysical and biochemical studies are performed. This difference has led both experimentalists and theoreticians… (more)

▼ The intracellular milieu differs from the dilute conditions in which most biophysical and biochemical studies are performed. This difference has led both experimentalists and theoreticians to tackle the challenging task of understanding how the intracellular environment affects the properties of biopolymers. Despite a growing number of in-cell studies, there is a lack of quantitative, residue-level information about equilibrium thermodynamic protein stability under non-perturbing conditions. My dissertation addresses this void by describing the use of NMR-detected hydrogen-deuterium exchange of quenched cell lysates to measure individual opening free energies of the 56-amino acid B1 domain of protein G (GB1) in living Escherichia coli cells without adding destabilizing co-solutes or heat. Comparisons to dilute solution data (pH 7.6 and 37 °C) show that opening free energies increase by as much as 1.14 ±0.05 kcal/mol in cells. Importantly, I also show that homogeneous protein crowders destabilize GB1, highlighting the challenge of recreating the cellular interior. These findings are discussed in terms of hard-core excluded volume effects, charge-charge GB1-crowder interactions and other factors. The quenched lysate method is applied further in mutational studies of GB1 to make the first quantification of non-specific protein-protein interactions in cells. I show that a surface mutation in GB1 is 10-times more destabilizing in E. coli than in buffer. The results indicate that quinary interactions between surface exposed residues and cytoplasmic proteins can play a key role in determining the native stability of a protein, whereas such a role is absent in buffer alone. The methods developed and applied throughout this work should prove useful for extension to other globular proteins in efforts to gain a more complete understanding of the effects of the intracellular environment on protein chemistry. Advisors/Committee Members: Monteith, William, Pielak, Gary J., Thompson, Nancy, Campbell, Sharon, Spremulli, Linda, Berkowitz, Max.

Subjects/Keywords: Chemistry; Biochemistry; Biophysics; College of Arts and Sciences; Department of Chemistry

Record Details Similar Records Cite « Share

❌

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Monteith, W. (2014). Residue Level Quantification of Protein Stability in Living Cells. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:2015b666-3d6f-4fce-98c8-d7f182ea4426

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Monteith, William. “Residue Level Quantification of Protein Stability in Living Cells.” 2014. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:2015b666-3d6f-4fce-98c8-d7f182ea4426.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Monteith, William. “Residue Level Quantification of Protein Stability in Living Cells.” 2014. Web. 18 Jan 2021.

Vancouver:

Monteith W. Residue Level Quantification of Protein Stability in Living Cells. [Internet] [Thesis]. University of North Carolina; 2014. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:2015b666-3d6f-4fce-98c8-d7f182ea4426.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Monteith W. Residue Level Quantification of Protein Stability in Living Cells. [Thesis]. University of North Carolina; 2014. Available from: https://cdr.lib.unc.edu/record/uuid:2015b666-3d6f-4fce-98c8-d7f182ea4426

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

18. Zigoneanu, Imola G. α-Synuclein Interaction with Membranes.

Degree: Chemistry, 2011, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:c46bcfc6-3d9d-446b-a2d9-bdc4680aa779

► The association of α-synuclein with membranes appears to be an important factor in Parkinson's disease. My dissertation research was focused on understanding the interactions of… (more)

Subjects/Keywords: College of Arts and Sciences; Department of Chemistry

Record Details Similar Records Cite « Share

❌

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Zigoneanu, I. G. (2011). α-Synuclein Interaction with Membranes. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:c46bcfc6-3d9d-446b-a2d9-bdc4680aa779

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Zigoneanu, Imola G. “α-Synuclein Interaction with Membranes.” 2011. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:c46bcfc6-3d9d-446b-a2d9-bdc4680aa779.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Zigoneanu, Imola G. “α-Synuclein Interaction with Membranes.” 2011. Web. 18 Jan 2021.

Vancouver:

Zigoneanu IG. α-Synuclein Interaction with Membranes. [Internet] [Thesis]. University of North Carolina; 2011. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:c46bcfc6-3d9d-446b-a2d9-bdc4680aa779.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Zigoneanu IG. α-Synuclein Interaction with Membranes. [Thesis]. University of North Carolina; 2011. Available from: https://cdr.lib.unc.edu/record/uuid:c46bcfc6-3d9d-446b-a2d9-bdc4680aa779

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

19. Tyrrell, Jillian. Effect of Macromolecular Crowding on the Structure and Function of a Riboswitch Aptamer in Cells and in Vitro.

Degree: Chemistry, 2013, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:2dc05712-7910-428d-b889-568b8b42ed04

► There are large differences between the cellular environment and the conditions widely used to study RNA structure and function in vitro, but until recently no… (more)

▼ There are large differences between the cellular environment and the conditions widely used to study RNA structure and function in vitro, but until recently no technologies allowed nucleotide-resolution analyses of RNA structure inside cells. We have now examined the structure, dynamics, and ligand-binding function of the adenine riboswitch aptamer domain in healthy, growing Escherichia coli cells at single-nucleotide resolution using SHAPE. We compared the in-cell RNA structure with that obtained in aqueous buffer containing 1 mM Mg2+, approximately the concentration that we directly measured inside cells. The fully folded ligand-bound aptamer formed essentially the same structure in cells as in buffer. In contrast, the unbound RNA aptamer in cells was much more highly structured than the ligand-free state in vitro. Even high in vitro concentrations of Mg2+ did not yield the degree of structural organization observed for the free aptamer in cells. The crowded cellular environment thus stabilizes, or pre-organizes, otherwise dynamic RNA conformations significantly more than does Mg2+ alone, demonstrating a profound influence of the cellular environment on RNA structure. To deduce how the structural effects observed in cells may be induced in vitro and how the aptamer ligand binding affinity is affected by crowding, studies in the presence of total cellular RNA, and the synthetic polymer, polyethylene glycol (PEG), were employed. These studies showed that RNA-RNA interactions and PEG had varying effects on aptamer structure. The structure of the aptamer in the presence of both cellular RNA and large PEGs agreed better with the structure observed in cells. However, specific characteristics observed for the aptamer in cells were not observed in vitro under either condition. Crowding the aptamer with PEG had little effect on ligand binding affinity, even when large structural effects were observed. These results suggest that the complex cellular environment is difficult to mimic in vitro, and to do so, may require a combination of crowding agents having different chemical properties. Finally, the observed minimal effects on ligand binding affinity for the aptamer in the presence of PEG, suggests that while RNA structure is affected by crowding in cells, RNA function may not be significantly affected. Advisors/Committee Members: Tyrrell, Jillian, Pielak, Gary J..

Subjects/Keywords: College of Arts and Sciences; Department of Chemistry

Record Details Similar Records Cite « Share

❌

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Tyrrell, J. (2013). Effect of Macromolecular Crowding on the Structure and Function of a Riboswitch Aptamer in Cells and in Vitro. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:2dc05712-7910-428d-b889-568b8b42ed04

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Tyrrell, Jillian. “Effect of Macromolecular Crowding on the Structure and Function of a Riboswitch Aptamer in Cells and in Vitro.” 2013. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:2dc05712-7910-428d-b889-568b8b42ed04.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Tyrrell, Jillian. “Effect of Macromolecular Crowding on the Structure and Function of a Riboswitch Aptamer in Cells and in Vitro.” 2013. Web. 18 Jan 2021.

Vancouver:

Tyrrell J. Effect of Macromolecular Crowding on the Structure and Function of a Riboswitch Aptamer in Cells and in Vitro. [Internet] [Thesis]. University of North Carolina; 2013. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:2dc05712-7910-428d-b889-568b8b42ed04.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Tyrrell J. Effect of Macromolecular Crowding on the Structure and Function of a Riboswitch Aptamer in Cells and in Vitro. [Thesis]. University of North Carolina; 2013. Available from: https://cdr.lib.unc.edu/record/uuid:2dc05712-7910-428d-b889-568b8b42ed04

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

20. Pollet, Rebecca. STRUCTURE AND FUNCTION STUDIES OF MICROBIAL CONJUGATIVE DNA TRANSFER & GI DRUG REACTIVATION PROCESSES.

Degree: Biochemistry and Biophysics, 2016, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:911890b6-9734-41d1-9e1b-b4eaf15ff4d9

► Antimicrobial resistance in Staphylococcus aureus presents an increasing threat to human health. This resistance is often encoded on mobile plasmids, such as pSK41; however, the… (more)

▼ Antimicrobial resistance in Staphylococcus aureus presents an increasing threat to human health. This resistance is often encoded on mobile plasmids, such as pSK41; however, the transfer mechanism of these plasmids is not well understood. In this study, we examine key protein features of the relaxase enzyme, NES, which initiates and terminates the transfer of the multidrug resistance plasmid pSK41. This work establishes that both a novel C-terminal domain and two loops of the NES protein, hairpin loops 1 and 2, are essential for proper DNA cleavage and religation by the full 665-residue NES protein in vitro. Second, we show that NES is able to bind, cleave, and religate the oriT sequences of non-conjugative plasmids pSK156 and pCA347. These data indicate that the conjugative relaxase in trans mechanism recently described for the pWBG749 family of plasmids also applies to the pSK41 family of plasmids, further heightening the potential significance of this mechanism in the horizontal transfer of staphylococcal plasmids. Finally, we use the knowledge of important NES features to design polyamide inhibitors that disrupt key protein-DNA interactions and chelator fragments that target a required coordinated metal ion. The efficacy of these inhibitors suggests that disrupting NES function may be a viable option for disrupting conjugative plasmid transfer. β-glucuronidase (GUS) enzymes are responsible for the severe drug toxicity associated with the chemotherapy drug irinotecan. Previous characterization of E. coli GUS identified inhibitors whose efficacy is dependent on interaction with an active site loop termed Loop 1. Analysis of the Human Microbiome Project sequencing data establishes a catalog of GUS sequences present in the human gastrointestinal tract. Sequences in this catalog are classified according to the presence and size of an active site loop and we identify at least one sequence from each class for further characterization. Characterization confirms the β-glucuronidase activity of seven new enzymes and establishes the ability of six GUS enzymes to process SN-38G. We also confirm the importance of pH for optimal enzyme function. This data expands current understanding of the β-glucuronidase enzyme family and provides additional GUS enzymes against which to optimize inhibitors to prevent SN-38G toxicity. Advisors/Committee Members: Pollet, Rebecca, Redinbo, Matthew R., Slep, Kevin, Pielak, Gary J., Neher, Saskia, Ke, Hengming, Richardson, Anthony.

Subjects/Keywords: School of Medicine; Department of Biochemistry and Biophysics

Record Details Similar Records Cite « Share

❌

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Pollet, R. (2016). STRUCTURE AND FUNCTION STUDIES OF MICROBIAL CONJUGATIVE DNA TRANSFER & GI DRUG REACTIVATION PROCESSES. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:911890b6-9734-41d1-9e1b-b4eaf15ff4d9

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Pollet, Rebecca. “STRUCTURE AND FUNCTION STUDIES OF MICROBIAL CONJUGATIVE DNA TRANSFER & GI DRUG REACTIVATION PROCESSES.” 2016. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:911890b6-9734-41d1-9e1b-b4eaf15ff4d9.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Pollet, Rebecca. “STRUCTURE AND FUNCTION STUDIES OF MICROBIAL CONJUGATIVE DNA TRANSFER & GI DRUG REACTIVATION PROCESSES.” 2016. Web. 18 Jan 2021.

Vancouver:

Pollet R. STRUCTURE AND FUNCTION STUDIES OF MICROBIAL CONJUGATIVE DNA TRANSFER & GI DRUG REACTIVATION PROCESSES. [Internet] [Thesis]. University of North Carolina; 2016. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:911890b6-9734-41d1-9e1b-b4eaf15ff4d9.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Pollet R. STRUCTURE AND FUNCTION STUDIES OF MICROBIAL CONJUGATIVE DNA TRANSFER & GI DRUG REACTIVATION PROCESSES. [Thesis]. University of North Carolina; 2016. Available from: https://cdr.lib.unc.edu/record/uuid:911890b6-9734-41d1-9e1b-b4eaf15ff4d9

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of North Carolina

21. Moore, Stephanie. Separation and Identification Techniques for Membrane Proteins using Ultra-High Pressure Liquid Chromatography coupled to Mass Spectrometry.

Degree: Chemistry, 2016, University of North Carolina

URL: https://cdr.lib.unc.edu/record/uuid:20ded5d0-3cf6-4491-8d7e-3d1315f03ec6

► Due to the importance of membrane proteins in biological pathways, the development of analytical techniques to improve membrane protein identifications is essential. For such complex… (more)

▼ Due to the importance of membrane proteins in biological pathways, the development of analytical techniques to improve membrane protein identifications is essential. For such complex mixtures, high resolution liquid chromatography (LC) is commonly utilized along with mass spectrometry (MS) for comprehensive proteomic analysis. However, commercial LC systems cannot provide the peak capacity required for such complex mixtures. With the advent of ultra-high pressure liquid chromatography (UHPLC) and multidimensional chromatography applications, peak capacities and protein identifications have increased. This dissertation will examine aspects of membrane protein sample preparation as well as instrumental analysis. Sample preparation techniques, specifically for membrane proteins, are crucial for proper protein analysis. Techniques involved with cell lysis, membrane protein extraction, solubilization, and digestion, are discussed (Chapter 2). Ultimately an optimized membrane protein sample preparation protocol was developed involving the use of high frequency sonication and the detergent sodium deoxycholate to improve solubilization. Peptides were ultimately analyzed on a modified (in-house) UHPLC constant pressure system. To improve proteomic separations, a new freeze/thaw valve and gradient storage loop were introduced to the UHPLC system, improving membrane protein identifications, instrumental reproducibility, and ruggedness (Chapter 3). To improve membrane protein digestion, an immobilized enzyme reactor (IMER) was introduced to the current multidimensional proteomic workflow. The IMER, placed after the protein separation, digested proteins as they eluted from the first dimension. This provided proteins with equivalent coverage to traditional in-solution digestions, yet avoided the 15 hr in-solution incubation process. Overall, the IMER produced high protein coverage (for both model and complex sample analysis) in a ~10 sec volumetric residence time on column (Chapter 4). Finally, a fully on-line two dimensional chromatographic system including IMER digestion was developed (Chapter 5). This system also provided equivalent coverage and protein identifications to in-solution off-line digestion, yet eliminated not only the overnight 15 hr digestion process as before, but also the ~4 hr fractionation and overnight lyophilization procedure from the workflow. Ultimately this saved the workflow about 35 hr and demonstrated the utility of the IMER. Advisors/Committee Members: Moore, Stephanie, Jorgenson, James, Schoenfisch, Mark H., Glish, Gary, Pielak, Gary J., Li, Bo.

Subjects/Keywords: College of Arts and Sciences; Department of Chemistry

Record Details Similar Records Cite « Share

❌

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Moore, S. (2016). Separation and Identification Techniques for Membrane Proteins using Ultra-High Pressure Liquid Chromatography coupled to Mass Spectrometry. (Thesis). University of North Carolina. Retrieved from https://cdr.lib.unc.edu/record/uuid:20ded5d0-3cf6-4491-8d7e-3d1315f03ec6

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Moore, Stephanie. “Separation and Identification Techniques for Membrane Proteins using Ultra-High Pressure Liquid Chromatography coupled to Mass Spectrometry.” 2016. Thesis, University of North Carolina. Accessed January 18, 2021. https://cdr.lib.unc.edu/record/uuid:20ded5d0-3cf6-4491-8d7e-3d1315f03ec6.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Moore, Stephanie. “Separation and Identification Techniques for Membrane Proteins using Ultra-High Pressure Liquid Chromatography coupled to Mass Spectrometry.” 2016. Web. 18 Jan 2021.

Vancouver:

Moore S. Separation and Identification Techniques for Membrane Proteins using Ultra-High Pressure Liquid Chromatography coupled to Mass Spectrometry. [Internet] [Thesis]. University of North Carolina; 2016. [cited 2021 Jan 18]. Available from: https://cdr.lib.unc.edu/record/uuid:20ded5d0-3cf6-4491-8d7e-3d1315f03ec6.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Moore S. Separation and Identification Techniques for Membrane Proteins using Ultra-High Pressure Liquid Chromatography coupled to Mass Spectrometry. [Thesis]. University of North Carolina; 2016. Available from: https://cdr.lib.unc.edu/record/uuid:20ded5d0-3cf6-4491-8d7e-3d1315f03ec6

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

		in
And / Or
		in
And / Or
		in
And / Or
		in

Open Access Theses and Dissertations