+publisher:"University of Colorado" +contributor:("Marcelo Sousa")

University of Colorado

1. LeBlanc, Marc-Andre. High Precision AFM-Based SMFS of Mechanically Labile Type III Secretion System Effectors.

Degree: PhD, 2018, University of Colorado

URL: https://scholar.colorado.edu/chem_gradetds/244

► Pathogenic bacteria have developed a wide range of tools for circumventing or overcoming the host’s defenses. Over time, these tools have become increasingly complex,… (more)

▼ Pathogenic bacteria have developed a wide range of tools for circumventing or overcoming the host’s defenses. Over time, these tools have become increasingly complex, allowing bacteria to live and thrive within a wide variety of host environments. One such tool is the Type III Secretion System (T3SS), a needle-like complex that allows bacteria to directly inject proteins, known as effectors, from their cytoplasm into host cells. Once inside host cells, effector proteins have a wide range of effects, from shutting down the host immune response to rearranging the host cytoskeleton to accommodate invading bacteria. Because the T3SS needle presents a narrow channel (< 2 nm), effector proteins must be mechanically unfolded before passing through. Proteins are unfolded by a molecular motor that associates with the base of needle and pulls protein into the channel. While this motor can unfold and secrete many proteins, it is unable to unfold proteins that have high mechanical stability. This indicates a need for effectors to be mechanically labile no matter their function. This may be one of the reasons effectors have very low sequence and structural similarity to other members of their protein super-families. This spurred our investigation into how effectors respond to mechanical force. To investigate effector protein stability, I used atomic force microscopy (AFM) to mechanically unfold the proteins. Here I show that effector proteins of the T3SS unfold at very low force, despite containing a wide variety folds and functions. This supports our hypothesis that to facilitate efficient secretion, effectors evolved to be mechanically labile. Because effector proteins unfold at such low force, it was critical for me to utilize site-specific attachment to both the AFM tip and surface, increasing both the amount of data I could collect and the quality of collected data. Site-specific attachment resulted in a 70-fold improvement in the yield of high quality data, allowing rapid characterization of mechanically labile α-helical proteins. Combining site-specific attachment with modified cantilevers allowed the collection of unfolding data for 5 effector proteins, finding they all unfold at low force (<20 pN), making them some of the most mechanically labile proteins studied to date by AFM-SMFS. Comparing the mechanical stability of effector proteins to their in vivo secretion rates, showed that unfolding force does not always correlate with in vivo secretion rate. However, the distance to the transition state does correlate with in vivo secretion rate. To elucidate how effector proteins have evolved to be efficiently secreted, the mechanical stability of an effector protein, NleC, was compared with a non-secreted homologue, protealysin. While the initial unfolding event of NleC occurs below the detection limit of our AFM platform, the unfolding of an intermediate along the unfolding pathway was measured. When compared to the unfolding of protealysin, the unfolding intermediate of… Advisors/Committee Members: Marcelo Sousa, Thomas Perkins, Michael Stowell, Joseph Falke, Amy Palmer.

Subjects/Keywords: afm; protein unfolding; single-molecule force spectroscopy; specific attachment; type iii secretion system; Biochemistry; Biophysics

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APA (6^th Edition):

LeBlanc, M. (2018). High Precision AFM-Based SMFS of Mechanically Labile Type III Secretion System Effectors. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/244

Chicago Manual of Style (16^th Edition):

LeBlanc, Marc-Andre. “High Precision AFM-Based SMFS of Mechanically Labile Type III Secretion System Effectors.” 2018. Doctoral Dissertation, University of Colorado. Accessed January 23, 2020. https://scholar.colorado.edu/chem_gradetds/244.

MLA Handbook (7^th Edition):

LeBlanc, Marc-Andre. “High Precision AFM-Based SMFS of Mechanically Labile Type III Secretion System Effectors.” 2018. Web. 23 Jan 2020.

Vancouver:

LeBlanc M. High Precision AFM-Based SMFS of Mechanically Labile Type III Secretion System Effectors. [Internet] [Doctoral dissertation]. University of Colorado; 2018. [cited 2020 Jan 23]. Available from: https://scholar.colorado.edu/chem_gradetds/244.

Council of Science Editors:

LeBlanc M. High Precision AFM-Based SMFS of Mechanically Labile Type III Secretion System Effectors. [Doctoral Dissertation]. University of Colorado; 2018. Available from: https://scholar.colorado.edu/chem_gradetds/244

University of Colorado

2. Manhart, Carol Michelle. Investigating protein-DNA interactions at replication forks by photo-crosslinking.

Degree: PhD, Chemistry & Biochemistry, 2013, University of Colorado

URL: https://scholar.colorado.edu/chem_gradetds/78

► During Okazaki fragment synthesis, the replicase must distinguish single-stranded from duplex DNA in advance of the polymerase to sense completion of a fragment and… (more)

▼ During Okazaki fragment synthesis, the replicase must distinguish single-stranded from duplex DNA in advance of the polymerase to sense completion of a fragment and trigger release from the lagging strand. A hypothesis in the literature proposes that the τ subunit (of the DnaX complex) directly senses completion of an Okazaki fragment. An alternative model suggests that the polymerase subunit senses conversion of a gap to a nick. I show using a novel phenyldiazirine photo-crosslinker linked to the 5-position of thymidylate that the τ subunit is not in position to distinguish gapped DNA from nicked DNA. The α subunit (the polymerase) is positioned to serve as the processivity sensor. Upon encountering duplex DNA, the polymerase likely changes conformation triggering its release from the lagging strand and the β processivity clamp, modulating its own affinity. Unrepaired replication forks dissociate from the helicase and suffer collapse. PriA recognizes stalled replication forks and initiates interactions to reload the helicase and activate a previously stalled fork. I used a FRET helicase assay to develop a PriA- dependent helicase loading system in E. coli and B. subtilis and to identify a minimal substrate to support a photo-crosslinking study also discussed here. I discovered that PriA's ATPase activity dictates substrate specificity. I also show that PriA serves as a checkpoint protein by blocking the replicase from binding to stalled replication forks distinguishing between an alternative model. SPP1 is a bacteriophage that infects B. subtilis. It encodes its own initiation proteins (origin binding protein, primosomal proteins, helicase, and single-strand binding protein (SSB)) but requires its host's primase and major replicative polymerase to replicate its genome. Both host and phage SSBs can support a reconstituted SPP1 system, but phage SSB does not support a reconstituted B. subtilis system. Using the B. subtilis FRET helicase assay, I show that phage SSB can substitute for the host's SSB in helicase reloading. Therefore the defect in the reconstituted system is not at the level of helicase loading or function and must occur after the helicase is loaded. I also show an absolute requirement on all SPP1 components in helicase reloading, including the origin binding protein (in a non-origin-containing template), which suggests a new role for this protein. In collaboration with Tim Lohman's lab at Washington University, I have contributed to a study into the functions of the C-terminal tails of SSB. SSB functions as a homotetramer whose four C-terminal tails interact with many other proteins necessary for DNA replication and repair. In an in vivo assay, an SSB variant that has two functional C-terminal tails supports viability in E. coli. An SSB variant that has one C- terminal tail is dominant lethal. In a reconstituted rolling circle E. coli replication system, there is a defect in coupled synthesis that causes a two-fold decrease in lagging strand synthesis relative to the leading strand using the variant… Advisors/Committee Members: Charles S. McHenry, Robert Kuchta, Robert Batey, Marcelo Sousa, Thomas Perkins.

Subjects/Keywords: Biochemistry

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

APA (6^th Edition):

Manhart, C. M. (2013). Investigating protein-DNA interactions at replication forks by photo-crosslinking. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/78

Chicago Manual of Style (16^th Edition):

Manhart, Carol Michelle. “Investigating protein-DNA interactions at replication forks by photo-crosslinking.” 2013. Doctoral Dissertation, University of Colorado. Accessed January 23, 2020. https://scholar.colorado.edu/chem_gradetds/78.

MLA Handbook (7^th Edition):

Manhart, Carol Michelle. “Investigating protein-DNA interactions at replication forks by photo-crosslinking.” 2013. Web. 23 Jan 2020.

Vancouver:

Manhart CM. Investigating protein-DNA interactions at replication forks by photo-crosslinking. [Internet] [Doctoral dissertation]. University of Colorado; 2013. [cited 2020 Jan 23]. Available from: https://scholar.colorado.edu/chem_gradetds/78.

Council of Science Editors:

Manhart CM. Investigating protein-DNA interactions at replication forks by photo-crosslinking. [Doctoral Dissertation]. University of Colorado; 2013. Available from: https://scholar.colorado.edu/chem_gradetds/78

University of Colorado

3. Hopkins, Alex Hunt. Toward a Mechanistic Understanding of Outer Membrane Protein Biogenesis.

Degree: PhD, Chemistry & Biochemistry, 2017, University of Colorado

URL: https://scholar.colorado.edu/chem_gradetds/220

► The outer membranes of Gram-negative bacteria are essential to their survival and have a unique asymmetric structure that features an outer leaflet of lipopolysaccharide. Bacterial… (more)

▼ The outer membranes of Gram-negative bacteria are essential to their survival and have a unique asymmetric structure that features an outer leaflet of lipopolysaccharide. Bacterial outer membranes are populated by integral membrane proteins with amphipathic transmembrane β-barrel domains called outer membrane proteins (OMPs). OMPs are translated in the cytoplasm and translocated across the inner membrane and periplasm before being inserted into the outer membrane. The β-barrel Assembly Machine (BAM), a hetero-pentameric protein complex at the outer membrane, recognizes, folds, and releases OMPs into the membrane. However, the functional contributions of individual BAM subunits, the architecture of the BAM, and the molecular mechanism by which the BAM recognizes and folds its substrates are unknown. To test whether the non-essential BAM subunits have OMP-specific functions or function generally in OMP biogenesis, the outer membrane proteomes of BAM subunit knockout strains were compared to wild-type via mass spectrometry. The general decrease of OMP abundance observed in the ΔbamB strain and very minor effects observed in the ΔbamC and ΔbamE strains suggest they play general roles in OMP biogenesis. To better understand the complex interface and provide a structural model of the entire BAM, the structural characterization of a BamA-BamD fusion protein was undertaken and the observed crystallographic interface validated in vivo. The model of BamABCD revealed a periplasmic ring formed by BamD and the POTRA domains of BamA that is proposed to bind nascent OMPs prior to membrane insertion. To enable experiments to analyze OMP biogenesis in vivo, OMP translocation intermediates were generated and characterized by periplasmic cysteine modification and OMP folding assays. To show that these intermediates can report on OMP biogenesis events in the periplasm, the intermediates were further characterized via site-specific photo-activatable cross-linking. The OMP translocation intermediates yielded site-specific cross-links that are modulated by both the location of the cross-linker in the β-barrel and the length of a linker domain that controls how far the β-barrel domain can extend across the periplasm. Therefore, the OMP translocation intermediates are a tool that can be used to track OMP biogenesis in vivo, test proposed OMP targeting mechanisms, and capture OMP folding intermediates. Advisors/Committee Members: Marcelo Sousa, Dylan Taatjes, Deborah Wuttke, Amy Palmer, Michael Stowell.

Subjects/Keywords: outer membrane lipopolysaccharide; inner membrane; protein; biogenesis; outer membrane protein; crosslinking; Biochemistry; Molecular Biology

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APA (6^th Edition):

Hopkins, A. H. (2017). Toward a Mechanistic Understanding of Outer Membrane Protein Biogenesis. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/220

Chicago Manual of Style (16^th Edition):

Hopkins, Alex Hunt. “Toward a Mechanistic Understanding of Outer Membrane Protein Biogenesis.” 2017. Doctoral Dissertation, University of Colorado. Accessed January 23, 2020. https://scholar.colorado.edu/chem_gradetds/220.

MLA Handbook (7^th Edition):

Hopkins, Alex Hunt. “Toward a Mechanistic Understanding of Outer Membrane Protein Biogenesis.” 2017. Web. 23 Jan 2020.

Vancouver:

Hopkins AH. Toward a Mechanistic Understanding of Outer Membrane Protein Biogenesis. [Internet] [Doctoral dissertation]. University of Colorado; 2017. [cited 2020 Jan 23]. Available from: https://scholar.colorado.edu/chem_gradetds/220.

Council of Science Editors:

Hopkins AH. Toward a Mechanistic Understanding of Outer Membrane Protein Biogenesis. [Doctoral Dissertation]. University of Colorado; 2017. Available from: https://scholar.colorado.edu/chem_gradetds/220

University of Colorado

4. Protter, David Stephen Warren. Contributions of Intrinsically Disordered Regions of Proteins to the Assembly of Ribonucleoprotein Granules.

Degree: PhD, Chemistry & Biochemistry, 2017, University of Colorado

URL: https://scholar.colorado.edu/chem_gradetds/242

► Cells assemble large, non-membrane bound granules of protein and RNA, termed Ri- bonucleoprotein granules (RNP granules), often in response to a wide variety of… (more)

Subjects/Keywords: intrinsically disordered region; phase separation; RNP granule; stress granule; Biochemistry

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

APA (6^th Edition):

Protter, D. S. W. (2017). Contributions of Intrinsically Disordered Regions of Proteins to the Assembly of Ribonucleoprotein Granules. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/242

Chicago Manual of Style (16^th Edition):

Protter, David Stephen Warren. “Contributions of Intrinsically Disordered Regions of Proteins to the Assembly of Ribonucleoprotein Granules.” 2017. Doctoral Dissertation, University of Colorado. Accessed January 23, 2020. https://scholar.colorado.edu/chem_gradetds/242.

MLA Handbook (7^th Edition):

Protter, David Stephen Warren. “Contributions of Intrinsically Disordered Regions of Proteins to the Assembly of Ribonucleoprotein Granules.” 2017. Web. 23 Jan 2020.

Vancouver:

Protter DSW. Contributions of Intrinsically Disordered Regions of Proteins to the Assembly of Ribonucleoprotein Granules. [Internet] [Doctoral dissertation]. University of Colorado; 2017. [cited 2020 Jan 23]. Available from: https://scholar.colorado.edu/chem_gradetds/242.

Council of Science Editors:

Protter DSW. Contributions of Intrinsically Disordered Regions of Proteins to the Assembly of Ribonucleoprotein Granules. [Doctoral Dissertation]. University of Colorado; 2017. Available from: https://scholar.colorado.edu/chem_gradetds/242

University of Colorado

5. Gifford, Jennifer Chappell. Interactions of Nanomaterials with Biological Systems: A Study of Bio-Mineralized Nanoparticles and Nanoparticle Antibiotics.

Degree: PhD, Chemistry & Biochemistry, 2015, University of Colorado

URL: https://scholar.colorado.edu/chem_gradetds/155

► Nature is continually able to out-perform laboratory syntheses of nanomaterials with control of specific properties under ambient temperatures, pressures and pH. The investigation of… (more)

▼ Nature is continually able to out-perform laboratory syntheses of nanomaterials with control of specific properties under ambient temperatures, pressures and pH. The investigation of existing biomolecule-mediated nanoparticle synthesis provides insight and knowledge necessary for duplicating these processes. In this way, peptides or proteins with nanomaterial mediation capabilities can be: 1) explored to further understand the ways in which biomolecules create specific nanoparticles then 2) used to create genetically encodable tags for use in electron tomography. The goal of designing such a tag was to assist in closing the resolution gap that exists in current imaging techniques between approximately 5 nm and 100 nm. Presented in this thesis are examples of peptides and proteins that form iron oxide, silver or gold nanoparticles under discrete circumstances. Three iron oxide-related bacterial proteins - bacterioferritin, Dps and Mms6 - were investigated for potential use. Similarly, a silver mineralizing peptide, Ge8, was studied upon attachment to the filamentous protein, FtsZ, and a gold mineralizing peptide, A3, was examined to characterize the way in which it mediates the formation of both Au⁰ nanoclusters and nanoparticles. Given the established interactions that occur between nanoparticles and biomolecules, it may not be surprising that gold nanoparticles displaying specific ratios of functional groups are able to interact with bacteria, in some cases inhibiting growth or causing cell death as antibiotics. A previously developed small molecule variable ligand display (SMVLD) method was expanded to identify a nanoparticle conjugate with a minimal inhibitory concentration (MIC_99.9) of 6 μM for Mycobacterium smegmatis, a common laboratory model for M. tuberculosis and the first example of SMVLD applied to mycobacteria. Nanoparticle structure-activity relationships, modes of action and approximations of mammalian cell toxicities were also explored to expand our understanding of how these nanoparticle antibiotics function and increase our ability to rationally design potential nanoparticle therapeutics for specific targets in the future. Finally, a new method for on-particle ligand quantitation via solid-state NMR spectroscopy was developed and applied to three different cases of nanoparticle conjugates. Advisors/Committee Members: Daniel L. Feldheim, Dylan Taatjes, Amy Palmer, Marcelo Sousa, Michael Stowell.

Subjects/Keywords: nanoparticle synthesis; mediation; genetically encodable tags; bacterial interaction; mammalian cell toxicity; on-particle ligand quantitation; Biochemistry; Cell Biology; Nanoscience and Nanotechnology

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

APA (6^th Edition):

Gifford, J. C. (2015). Interactions of Nanomaterials with Biological Systems: A Study of Bio-Mineralized Nanoparticles and Nanoparticle Antibiotics. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/155

Chicago Manual of Style (16^th Edition):

Gifford, Jennifer Chappell. “Interactions of Nanomaterials with Biological Systems: A Study of Bio-Mineralized Nanoparticles and Nanoparticle Antibiotics.” 2015. Doctoral Dissertation, University of Colorado. Accessed January 23, 2020. https://scholar.colorado.edu/chem_gradetds/155.

MLA Handbook (7^th Edition):

Gifford, Jennifer Chappell. “Interactions of Nanomaterials with Biological Systems: A Study of Bio-Mineralized Nanoparticles and Nanoparticle Antibiotics.” 2015. Web. 23 Jan 2020.

Vancouver:

Gifford JC. Interactions of Nanomaterials with Biological Systems: A Study of Bio-Mineralized Nanoparticles and Nanoparticle Antibiotics. [Internet] [Doctoral dissertation]. University of Colorado; 2015. [cited 2020 Jan 23]. Available from: https://scholar.colorado.edu/chem_gradetds/155.

Council of Science Editors:

Gifford JC. Interactions of Nanomaterials with Biological Systems: A Study of Bio-Mineralized Nanoparticles and Nanoparticle Antibiotics. [Doctoral Dissertation]. University of Colorado; 2015. Available from: https://scholar.colorado.edu/chem_gradetds/155

University of Colorado

6. Xiao, Yao. Conformational Dynamics in the Regulation of MAP Kinase, ERK2.

Degree: PhD, Chemistry & Biochemistry, 2015, University of Colorado

URL: https://scholar.colorado.edu/chem_gradetds/168

► The MAP kinase, extracellular signal-regulated kinase 2 (ERK2), is a key regulator of cell signaling. Aberrant up-regulation of ERK2 has been correlated with various… (more)

▼ The MAP kinase, extracellular signal-regulated kinase 2 (ERK2), is a key regulator of cell signaling. Aberrant up-regulation of ERK2 has been correlated with various diseases. ERK2 can be activated by MAP kinase kinases through dual phosphorylation at the activation loop. It remains a challenging question how changes in conformational dynamics contribute to kinase activation. NMR relaxation dispersion experiments were used to monitor changes in Ile, Leu, and Val (ILV) methyl motions in microsecond-millisecond timescale upon activation of ERK2. A structure-based procedure was developed to assign 13C1H3-labeled methyls, by comparing NMR distance constraints with the X-ray structure. This procedure yielded 60% of the methyl assignments in inactive and active forms of ILV 13C1H3-methyl labeled ERK2. In inactive ERK2, localized conformational dynamics was observed among methyls. Upon activation, the dynamics of assigned methyls in ERK2 were altered throughout the kinase core, including many residues in the catalytic pocket. The majority of methyls in active ERK2 fit to a single conformational exchange process, suggesting global domain motions involving interconversion between two states. A mutant of ERK2, engineered to enhance flexibility at the hinge region linking the N- and C-terminal domains, induced two-state conformational exchange throughout the kinase core. A mono-phospho-mimetic form of this mutant showed 25% of the dual-phosphorylated ERK2 activity. Thus, activation of ERK2 leads to a dramatic shift in conformational exchange, from a “tense” (T) state to a “relaxed” (R) state, likely through release of constraints at the hinge. To understand the effects on the conformational dynamics of ERK2 during catalysis and upon inhibitor binding, complexes of ERK2 with various ligands were formed. The binding of nucleotides and/or peptide substrates showed no significant perturbation to the T/R conformational equilibrium, with small enhancement of the T state population in active ERK2. In addition, differential conformational stabilization effects, which were not previously reported for ERK2, were observed upon the binding of different tight-binding inhibitors of ERK2. This thesis reports that ERK2 activation enhances microsecond-millisecond interconversion between conformers underlying different enzyme intermediates, thus linking protein dynamics to the catalytic cycle. The perturbations of conformational equilibrium by inhibitors reflect a novel allosteric mechanism in ERK2. Advisors/Committee Members: Natalie G. Ahn, Arthur Pardi, Marcelo Sousa, Johannes Rudolph, Loren Hough.

Subjects/Keywords: ERK2; MAP kinase; aberrant up-regulation; interconversion; conformation exchange; Biochemistry; Cell Biology

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

APA (6^th Edition):

Xiao, Y. (2015). Conformational Dynamics in the Regulation of MAP Kinase, ERK2. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/168

Chicago Manual of Style (16^th Edition):

Xiao, Yao. “Conformational Dynamics in the Regulation of MAP Kinase, ERK2.” 2015. Doctoral Dissertation, University of Colorado. Accessed January 23, 2020. https://scholar.colorado.edu/chem_gradetds/168.

MLA Handbook (7^th Edition):

Xiao, Yao. “Conformational Dynamics in the Regulation of MAP Kinase, ERK2.” 2015. Web. 23 Jan 2020.

Vancouver:

Xiao Y. Conformational Dynamics in the Regulation of MAP Kinase, ERK2. [Internet] [Doctoral dissertation]. University of Colorado; 2015. [cited 2020 Jan 23]. Available from: https://scholar.colorado.edu/chem_gradetds/168.

Council of Science Editors:

Xiao Y. Conformational Dynamics in the Regulation of MAP Kinase, ERK2. [Doctoral Dissertation]. University of Colorado; 2015. Available from: https://scholar.colorado.edu/chem_gradetds/168

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