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Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry and Biophysics, 2020., Anti-HIV broadly neutralizing antibodies (bNAbs) inactivate multiple strains of HIV by binding to the HIV envelope protein (Env), which consists of gp120 and gp41 subunits. However, immunization strategies based on the envelope protein have not been successful in eliciting anti-HIV bNAbs. This is at least partially explained by the fact that the envelope protein generally binds poorly to predicted germline-encoded precursor forms of bNAbs that must bind antigen in order to mature. To identify variant forms of Env with increased affinities for the precursor forms of bNAbs, we express libraries of randomly mutated HIV envelope proteins in a yeast surface display system and use fluorescence activated cell sorting to identify cells with enhanced abilities to bind the bNAb precursors. Based on analysis of individual sorted clones and on next generation sequencing of sorted libraries, we have identified mutations that significantly enhance the affinity of binding of Env to bNAbs directed against the Membrane Proximal External Region (MPER) of gp41. These mutations are enriched for substitutions of arginine for strongly conserved tryptophan residues in gp41 that reside outside the known epitopes for mature forms of these BNAbs, but map to critical contacts in structures of trimeric assemblies of Env.

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry and Biophysics, 2015., It has been generally assumed that signaling responses to an agonist are proportional to the number of receptors occupied on the cell surface. On studying this theory in the well-characterized yeast mating pathway, we observed anomalous relationships between receptor occupancy and signaling responses that are not readily explained by our current knowledge of receptor signaling. Ste2p is a G protein coupled receptor that activates the yeast mating pathway upon binding to its agonist, a-factor. Detailed quantitative analyses of signaling responses to agonist show that the EC50 (half maximal effective concentration) does not vary significantly over a wide range of receptor expression levels (~100-fold). Several a-factor analogs act as antagonists toward Ste2p, exhibiting high affinity binding to receptors, but eliciting minimal signaling responses. We find that antagonist competition for agonist signaling, despite effective competition for binding, is much weaker (>30-fold) than would be predicted based on the measured binding affinities of the respective ligands. We show that this discrepancy is not due to the different assay conditions used to measure binding and signaling, or the preferential association of receptor with downstream components such as G proteins or Sst2p (regulator of G protein signaling). In order to study the effect of signaling by partially-occupied oligomerized receptors, we have screened for potential oligomerization defective mutant alleles of Ste2p, using a novel fluorescence based screen, also discussed in this thesis. Another unexpected aspect of signaling response in the pheromone pathway is based on microscopy of fluorescently labeled antagonists. We find that antagonist-bound receptors are internalized at rates that are similar to, or faster than, the rate of internalization of agonist-bound receptors. This suggests biased agonism by the antagonist, which triggers internalization of the receptor without activating the signaling pathway. T

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry, 2013., Membrane proteins play integral roles in many cellular processes, including cell signaling, homeostasis, and cell adhesion. However, structures of membrane proteins are disproportionately underrepresented in databases like the Protein Data Bank (PDB). One proposed reason for the difficulty in determining the structures of membrane proteins is their instability, especially when solubilized in detergent. This idea is supported by several strategies for protein stabilization that have recently been used to determine structures of G Protein Coupled Receptors (GPCRs). In order to identify the poorly-understood determinants of membrane protein stability, and as a step toward structure determination, we have developed several screens to identify mutations that modulate the stability of Ste2p, a GPCR in the yeast mating pathway. Our overall goal was to identify thermally stabilized variant forms of Ste2p which contain mutations that allow the protein to maintain its native conformation at elevated temperatures. Since the thermal denaturation of normal Ste2p occurs at temperatures that are high enough to cause general detrimental effects to the organism, we initially identified temperature sensitive alleles that could be screened for second-site intragenic suppressors of the temperature sensitive phenotype. Using two different screens based on receptor signaling and receptor surface expression, we have identified seven new temperature-sensitive alleles of STE2 containing single amino acid substitutions and additional alleles containing multiple amino acid substitutions. In most cases, we found that the cellular secretion apparatus efficiently degrades destabilized receptors. Starting with several different temperature-sensitive alleles, we used Fluorescence Activated Cell Sorting (FACS) in conjunction with fluorescently labeled ligand to screen libraries of randomly mutated receptors for second site mutations that result in preservation of native receptor conformation at hig

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry and Biophysics, 2012., OxlT, an oxalate/formate transporter from the bacterium Oxalobacter formigenes is a member of the widely distributed class of Major Facilitator Superfamily (MFS) of transport proteins that play an important role in regulation of oxalate in the human intestinal tract. This thesis describes studies of the functional mechanism, oligomeric state, folding, and stability of OxlT as an example of this important class of MFS transporters. Using triple-detector size exclusion chromatography with static light scattering (SEC-LS), we showed that OxlT is a monomer in solution when solubilized by a variety of detergents. OxlT also failed to crosslink through disulfides or through chemical crosslinkers both in solution and in the membrane. Finally, functional reconstitution of OxlT in proteoliposomes at low protein:lipid ratios confirmed that the protein retained the functional monomeric state in the membrane as well. The folding and stability of membrane proteins is currently only poorly understood. We have employed several different spectroscopic techniques for understanding the folding and unfolding pathways of OxlT. Changes in intrinsic tryptophan fluorescence of OxlT showed a biphasic change upon addition of the denaturant SDS, indicative of the presence of a partially folded intermediate. We also developed protocols to label OxlT with Cy3-MTSEA and Cy5-MTSEA dyes that have provided significant enhancement of specificity of labeling compared to routinely used maleimide based dyes. Based on measurements of Fluorescence Resonance Energy Transfer (FRET), doubly labeled cysteine mutants of OxlT (F18C-A342C and G196C-A342C) also showed a biphasic decrease in energy transfer in the presence of increasing concentrations of SDS. Our experiments also suggested that OxlT unfolding is at least partially reversible. Initial single molecule FRET measurements of OxlT under conditions of free diffusion are also indicative of the presence of a folding intermediate. We have also documented F

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry and Molecular Biology, 2010., Birt-Hogg-Dubé syndrome (BHD) is a hereditary human cancer disorder characterized by renal carcinoma of diverse histology, benign skin lesions and pulmonary cysts, and is caused by mutations in the tumor suppressor gene BHD, which encodes folliculin (FLCN). The biological functions of BHD, however, remain unknown. Here we show that the Drosophila homolog of BHD localizes to the nucleolus and physically interacts with the 19S proteasomal ATPase, Rpt4, a nucleolar resident and known regulator of rRNA gene transcription that associates with the ribosomal DNA (rDNA) locus. Upon expression of BHD, Rpt4 binding to the rDNA locus was abolished, accompanied by a concomitant decrease in pre-rRNA levels. Down-regulation of BHD resulted in increased nucleolar volume and upregulation of rRNA synthesis, while BHD overexpression reduced rRNA transcription and counteracted the effects of Rpt4 over-expression on rRNA production, possibly by preventing association of Rpt4 with the rDNA locus. These changes in rRNA levels affected animal growth, and RasV12-induced tumor phenotypes. Our study suggests that BHD, by interacting with Rpt4, plays a role in controlling rRNA synthesis, which may be an important mechanism contributing to the pathology and progression of renal tumorigenesis in individuals afflicted with BHD syndrome.