The organization of two or more biological macromolecules into a functioning assembly is critical for many biological functions to occur. This phenomenon is the result of subtle interplay between complimentary structural and electrostatic factors. While a growing protein data bank of solved protein structures provides experimental evidence for studying the structural factors that stabilize protein-protein interface, there has been little advance in experimental determination of the electrostatic contributions. This lack of experimental investigation into protein electrostatics results in an inability to describe or predict how protein-protein complexes are arranged and stabilized. This problem is addressed in this dissertation by use of vibrational Stark effect (VSE) spectroscopy in which the spectral transitions of a vibrational probe are directly related to the strength and direction of the electric fields in the vicinity of the probe. The work presented here details an approach using VSE spectroscopy coupled with molecular dynamics simulation (MD) to interpret the role that electrostatics play in organizing the signaling protein Ras' interactions with its downstream effectors Raf and Ral guanosine dissociation simulator (RalGDS). Each chapter describes a specific set of experiments and MD simulations designed to understand the nature of protein-protein interactions. In Chapter 3, changes in the absorption energy of the nitrile probe at nine positions along the Ras-Ral interface were compared to results of a previous study examining this interface with Ral-based probes, and a pattern of low electrostatic field in the core of the interface surrounded by a ring of high electrostatic field around the perimeter of the interface was found. The areas of conserved Stark shifts are used to help describe electrostatic factors that stabilize the Ras-Ral interface. In Chapter 4, VSE is used to describe an electrostatic origin to the binding tilt between complexes formed between Ras and its two effectors Raf and Ral. There are three regions of conserved Stark effect shifts upon docking with WT Ras between the two effectors, indicating that the docked complexes conserve electrostatic fields, resulting in different binding orientation of otherwise structurally similar proteins. Chapter 5 details the use of MD simulation in correlation with VSE data for 18 mutants of the Ras at the oncogenic position 61 site. The combination of experimental and simulations support the hypothesis that position 61 on Ras is used to coordinate an active site water molecule during native guanosine triphosphate (GTP) hydrolysis. / text

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Ovarian cancer is a leading cause of death among of gynecological cancers. Current therapies are ineffective with a poor 5-year survival of only ~25%. p70 S6 kinase (p70 S6K) is a downstream target of the phosphatidylinositol 3-kinase pathway and is frequently activated in human ovarian cancer. However, the molecular targets and signaling pathways by which p70 S6K may affect tumor development and progression are poorly understood. Interestingly, in the laboratory, Mdm2, an important negative regulator of the p53 tumor suppressor, was identified in a yeast two hybrid screening of potential interacting partners for p70 S6K. In this study, I aimed to investigate the specific interaction of p70 S6K and Mdm2 and determine how this may contribute to ovarian tumorigenesis. Using a co-immunoprecipitation assay, the in vivo interaction of p70 S6K and Mdm2 in human ovarian cancer cells was confirmed. Upon UV-induced genotoxic stress, p70 S6K activation was associated with Mdm2 phosphorylation on S166 and subsequent p53 accumulation. This could be reversed by the use of rapamycin and p70 S6K siRNA to inhibit its kinase activity and expression respectively, confirming that the effect was p70 S6K specific. Conversely, ectopic expression of wildtype p70 S6K or a constitutively active mutant of p70 S6K, D3E-E389 (D3E) was sufficient to induce phosphorylation of Mdm2. Moreover, the p70 S6K mediated activation of Mdm2 was independent of p53 mutations. Similar results were observed upon other stress challenges such as hypoxia using hypoxia mimicking agent desferrioxamine (DFX). These findings identify Mdm2 as a new target of p70 S6K and reveal that p70 S6K intervenes the Mdm2-p53 regulatory loop in ovarian cancer, which may provide a survival advantage to cancer cells under stress conditions. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

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Not available / text

The discovery of the crucial intermediates and pathway in the process of the reverse transcription was reported using single-molecule spectroscopy and related techniques including single-molecule fluorescence resonance energy transfer, fluorescence correlation spectroscopy and confocal imaging. Reverse transcription of the HIV-1 RNA genome involves several complex nucleic acid rearrangement steps that are catalyzed by the HIV-1 nucleocapsid protein (NC), including for example, the annealing of the transactivation response (TAR) region of the viral RNA to the complementary region (TAR DNA) in minus-strand strong-stop DNA. In this dissertation, the research focused on elucidating the mechanism of NC-facilitated TAR DNA/RNA annealing. The single molecule spectroscopic measurements reported that the crucial intermediates as well as the mechanistic insight into the annealing of TAR RNA with TAR DNA mediated by viral NC proteins. The data reveal that NC partially melted the secondary structure of TAR DNA (termed the "YTAR") as well as TAR RNA. In the subsequent studies, various short DNA oligonucleotdies were applied to anneal with the TAR to mimic the initial annealing steps. The data support that the YTAR serves as a nucleation center for the annealing to occur through the multiple sites along the TAR structure. Two major nucleation pathways were observed, which are the annealing through the 3'/5' termini, namely "zipper" pathway and the annealing through the hairpin loop region, namely "kissing" pathway. The annealing mechanism was further explored by performing the annealing of wild-type TAR DNA with wild-type TAR RNA in the presence of NC in vitro. The annealing kinetic data suggest that the nucleation of TAR DNA/RNA annealing occurs in an encounter complex form in which one or two DNA/RNA strands in the "Y" form associated with multiple NC molecules. This encounter complex leads to the multiple nucleation complexes, i.e. zipper or kissing intermediates. The data further indicate that although the two complementary strands nucleate at multiple sites, i.e. any single-strand region of TAR, the annealing of two TAR complements occurs through a common mechanism.

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Protein-protein interactions are central to their biological functions in cells. Many approaches have been applied to study protein-protein interactions in a genomic-scale. In an attempt to develop new strategies to study protein-protein interactions, FRET by using ECFP and EYFP as the donor and receptor was evaluated for possible application in protein-protein interaction study in a high-throughput fashion. Due to the intrinsic properties of ECFP and EYFP, FRET-based protein-protein interaction assay is not suitable for large-scale studies. Instead, tandem affinity purification coupled with mass spectrometry approach proved to be a useful strategy to identify protein interacting partners. Several transcription factor complexes in yeast were successfully purified and novel components in the complexes were identified by combining a shotgun mass spectrometry approach and a differential analysis of the mass spectrometry data. In particular, a negative regulator of G1 to S phase transition during cell cycle, Whi5p, was identified to be a component of SBF complex; a regulator of nitrogen metabolism, Gln3p, was identified to be a component of Hap2/3/5 complex that regulates carbon metabolism, suggesting a crosstalk between nitrogen and carbon metabolism. Additionally, one-step purification coupled with shotgun mass spectrometry analysis was applied to simplify and improve the affinity purification approach used for protein-protein interaction studies. In order to map protein complexes in their native state, a sucrose density gradient was used to separate protein complexes in cells. The proteins within each fraction from the sucrose density gradient were analyzed and quantified with mass spectrometry to obtain the protein abundance profiles across the gradient. The known protein complexes were identified by clustering the protein abundance profiles. This method could possibly be improved to become a generic approach to mapping protein complexes. The goal of protein-protein interaction studies is to determine the protein functions. In an effort to identify ribosome biogenesis genes from a yeast gene network reconstructed from diverse large-scale interaction data sets, at least 25 new ribosome biogenesis genes were confirmed by extensive experimental validations, underscoring the value of proteinprotein interaction studies and gene interaction network.

Genetic mapping of complex traits has been successful over the last decade, with over 2,000 regions in the genome associated to disease. Yet, the translation of these findings into a better understanding of disease biology is not straightforward. The true promise of human genetics lies in its ability to explain disease etiology, and the need to translate genetic findings into a better understanding of biological processes is of great relevance to the community. We hypothesized that integrating genetics and protein- protein interaction (PPI) networks would shed light on the relationship among genes associated to complex traits, ultimately to help guide understanding of disease biology. First, we discuss the design, testing and implementation of a novel in silico approach (“DAPPLE”) to rigorously ask whether loci associated to complex traits code for proteins that form significantly connected networks. Using a high-confidence set of publically available physical interactions, we show that loci associated to autoimmune diseases code for proteins that assemble into significantly connected networks and that these networks are predictive of new genetic variants associated to the phenotypes in question. Next, we study variation in the electrocardiographic QT-interval, a heritable phenotype that when prolonged is a risk factor for cardiac arrhythmia and sudden cardiac death. We show that a large proportion of QT-associated loci encode proteins that are members of complexes identified by immunoprecipitations in mouse cardiac tissue of proteins known to be causal of Mendelian long-QT syndrome. For several of the identified proteins, we show they affect cardiac ion channel currents in model organisms. Using replication genotyping in 17,500 individuals, we use the complexes to identify genome-wide significant loci that would have otherwise been missed. Finally, we consider whether PPIs can be used to interpret rare and de novo variation discovered through recent technological advances in exome-sequencing. We report a highly connected network underlying de novo variants discovered in an autism trio exome-sequencing effort, and we design, test and implement a novel statistical framework (“DAPPLE/SEQ”) to analyze rare inherited variants in the context of PPIs in a way that significantly boosts power to detect association.