Understanding how proteins fold and bind is interesting since these processes are central to most biological activity. Protein folding and protein-protein interaction are by themselves very complex but using a good and robust system to study them could ease some of the hurdles. In this thesis I have tried to answer some of the fundamental questions of protein folding and binding. I chose to work with PDZ domains, which are protein domains consisting of 90-100 amino acids. They are found in more than 400 human proteins and function mostly as protein-protein interaction units. These proteins are very stable, easy to express and purify and their folding reaction is reversible under most laboratory conditions. I have characterized the interaction of PSD-95 PDZ3 domain with its putative ligand under different experimental conditions and found out that its binding kinetics is sensitive to salt and pH.  I also demonstrated that the two conserved residues R318 and H372 in PDZ3 are responsible for the salt and pH effect, respectively, on the binding reaction. Moreover, I determined that for PSD 95 PDZ3 coupling of distal residues to peptide binding was better described by a distance relationship and there was a very weak evidence of an allosteric network. Further, I showed that another PDZ domain, SAP97 PDZ2 undergoes conformational change upon ligand binding. Also, I characterized the binding mechanism of a dimeirc ligand/PDZ1-2 tandem interaction and showed that despite its apparent complexity the binding reaction is best described by a square scheme. Additionally, I determined that for the SAP 97 PDZ/HPV E6 interaction that all three PDZ domains each bind one molecule of the E6 protein and that a set of residues in the PDZ2 of SAP 97 could operate in an unexpected long-range manner during E6 interaction. Finally, I showed that perhaps all members in the PDZ family could fold via a three state folding mechanism. I characterized the folding mechanism of five different PDZ domains having similar overall fold but different primary structure and the results indicate that all five fold via an intermediate with two transition states. Transition state one is rate limiting at low denaturant concentration and vice versa for transition state two. Comparing and characterizing the structures of the transition states of two PDZ domains using phi value analysis indicated that their early transition states are less similar as compared to their late transition states.

Immediately following a gene duplication event, if both gene copies are to be fixed into a species’ genome there is a period of enhanced selection acting on either one or both duplicates (paralogs) that results in some extent of functional divergence. However, as redundancy among extant duplicates is thought to confer genomic robustness, a consequent question is: how much functional overlap exists between duplicates that are retained over long spans of evolutionary time? To examine this issue I determined the extent of shared protein interactions and protein complex membership for paralogous gene pairs resulting from an ancient Whole Genome Duplication (WGD) event in yeast, finding retained functional overlap to be substantial among this group. Surprisingly however, I found paralogs existing within the same complex tended to maintain greater disparities in expression, suggesting the existence of previously proposed “transcriptional back-up” mechanisms. To test both for existence of such mechanisms and for any phenotypic manifestation of their shared functional overlap I surveyed for the presence of aggravating genetic interactions between 399 WGD-resultant paralog pairs. While these paralogs exhibited a high frequency (~30%) of epistasis, observed genetic interactions were not predictable based on protein interaction overlap. Further, exposure to a limited number of stressors confirmed that additional instances of epistasis were only observable under alternate conditions. As only a small number of stress conditions were tested, the high frequency of genetic interactions reported appears to be a minimum estimate of the true extent of epistasis among WGD paralogs, potentially explaining the lack of overlap with protein interaction data. As it is impossible to survey an infinite condition space, Synthetic Genetic Array (SGA) screening of yeast strains carrying double-deletions of paralog pairs was used to assess functional redundancy among a group of the remaining non-epistatic paralog pairs. The resulting interactions demonstrated functional relationships in non-epistatic paralogs only obvious upon ablation of both duplicates, suggesting that these interactions had initially been masked through redundant function. These findings ultimately suggest an advantage to retained functional overlap among whole genome duplicates that is capable of being stably maintained through millions of years of evolutionary time.

<p> Bacterial cell division is an essential process, which is initiated by forming the Z-ring as a cytoskeletal scaffold at the midcell site, followed by the recruitment of a series of divisome proteins. In <i>Escherichia coli</i> (Ec), at least 15 divisome proteins (FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsB, FtsL, FtsI, FtsW, FtsN, FtsE, FtsX, ZapA, AmiC, EnvC) have been implicated in this process. The components of the cell division machinery proteins in <i>Neisseria gonorrhoeae</i> (Ng) differs from <i>E. coli. N. gonorrhoeae</i> possesses FtsA, but lacks FtsB. ZipA and FtsL in <i>N. gonorrhoeae</i> have low identity to ZipA and FtsL from <i>E. coli</i>. Our laboratory has studied the central division protein FtsZ in <i>N. gonorrhoeae</i>. Thus, my research investigated the role of <i>N. gonorrhoeae</i> FtsA in cell division and investigated the interactions between divisome proteins from <i>N. gonorrhoeae</i> to understand divisome assembly.</p> <p>This study determined the association of FtsA_Ng with FtsZ</sub>Ng and other divisome proteins in <i>N. gonorrhoeae</i> and identified the functional domains of FtsA<sun>Ng</sub> involved in these interactions using a bacterial two-hybrid (B2H) assay. FtsA_Ng interacted with FtsZ_Ng, FtsK_Ng, FtsW_Ng, FtsQ_Ng, and FtsN_Ng. Self-interactions of FtsA_Ng and FtsZ_Ng were also detected. FtsI_Ng, FtsE_Ng and FtsX_Ng did not interact with FtsA_Ng. The 2A₁, 2A₂ and 2B domains of FtsA_Ng were sufficient to interact with FtsZ_Ng independently. Domain 2A₁ interacted with FtsK_Ng and FtsN_Ng. Domain 2B of FtsA_Ng interacted with FtsK_Ng, FtsQ_Ng, and FtsN_Ng. Domain 2A₂ of FtsA_Ng interacted with FtsQ_Ng, FtsW_Ng, and FtsN_Ng. These data suggest that FtsA in <i>N. gonorrhoeae</i> plays a key role in interactions with FtsZ and other divisome proteins.</p> <p>The potential interactions between divisome proteins in <i>N. gonorrhoeae</i> were examined using B2H assays. The comparisons between the <i>N. gonorrhoeae</i> divisome protein interaction network and those of <i>E. coli</i> and <i>S. pneumoniae</i> indicates that the divisome protein interactome of <i>N. gonorrhoeae</i> is more similar to that of <i>S. pneumoniae</i> and differs from that of <i>E. coli</i>. The comparisons revealed that compared to the interactions in <i>E. coli</i> and <i>S. pneumoniae</i>, more interactions between divisome proteins upstream of FtsA_Ng (including FtsA_Ng) and downstream of FtsA_Ng were observed in <i>N. gonorrhoeae</i> while fewer interactions between divisome proteins downstream of FtsA_Ng were observed in <i>N. gonorrhoeae</i>. Possible reasons for this include the inability of ZipA_Ng to interact with other divisome proteins and the absence of FtsL and FtsB in <i>N. gonorrhoeae</i>, resulting in the lack of an FtsQ-FtsB-FtsL complex in <i>N. gonorrhoeae</i>. These results indicate a possibly different divisome assembly in <i>N. gonorrhoeae</i> from that proposed models for <i>E. coli</i>.</p> A model for FtsA_Ng structure was predicted based on structural homology modeling with the resolved crystal structure of <i>Thermotoga maritima</i> FtsA. Four domains on the molecule were identified, designated 1A, 1C, 2B and 2A (including 2A₁ and 2A₂). Domains 2A and 2B of FtsA were highly conserved based on multi-sequence alignments of FtsAs from 30 bacteria. FtsA_Ng located to the division site in <i>N. gonorrhoeae</i> cells and the ratio of FtsA to FtsZ ranged from 1:24 to 1: 33 in three <i>N. gonorrhoeae</i> strains, which gave a lower cellular concentration of FtsA compared to other organisms.</p> <p>I also determined that overexpression of FtsA_Ng in <i>E. coli</i> led to cell filamentous in rod-shaped <i>E. coli</i> and cell enlargement and aggregation in mutant, round <i>E. coli</i>. FtsA_Ng failed to complement an <i>ftsA</i>_Ec-deletion <i>E. coli</i> strain although the overexperssion of FtsA_Ng disrupted <i>E. coli</i> cell division. In addition, overexpression of FtsA_Ng only affected cell division in some cells and its localization in <i>E. coli</i> was independent of interaction with <i>E. coli</i> FtsA or FtsZ. These results indicate that FtsA_Ng exhibits a species-specific functionality and <i>E. coli</i> is not a suitable model for studying FtsA_Ng functionality.</p> <p>This is the first study to characterize FtsA from <i>N. gonorrhoeae</i> in cell division. I identified novel functional domains of FtsA_Ng involved in interactions with other divisome proteins. The <i>N. gonorrhoeae</i> divisome protein interaction network determined by B2H assays provides insight into divisome assembly in <i>N. gonorrhoeae</i></p>.

The Membrane Yeast Two-Hybrid (MYTH) technology was used in this study to find protein-protein interactors of Saccharomyces cerevisiae ATP binding cassette (ABC) transporters Nft1p, Pdr10p, Pdr18p and Vmr1p. There were 23 interactors for Nft1p, 22 interactors for Pdr10p, 4 interactors for Pdr18p and 1 interactor for Vmr1p. The 43 unique interactors belong to a wide variety of functional categories. There were 11 interactors involved in metabolism, 9 interactors involved in transport, 8 interactors with unknown function, 4 interactors involved in trafficking and secretion, 3 interactors involved in protein folding, 2 interactors involved in stress response, and 1 interactor in each of the following categories: cell wall assembly, cytoskeleton maintenance, nuclear function, protein degradation, protein modification and protein synthesis. Follow up experiments also showed that Pdr15p and Pdr18p play an important role in zinc homeostasis because deletion of these ABC transporters results in sensitivity to zinc shock.

The Membrane Yeast Two-Hybrid (MYTH) technology was used in this study to find protein-protein interactors of Saccharomyces cerevisiae ATP binding cassette (ABC) transporters Nft1p, Pdr10p, Pdr18p and Vmr1p. There were 23 interactors for Nft1p, 22 interactors for Pdr10p, 4 interactors for Pdr18p and 1 interactor for Vmr1p. The 43 unique interactors belong to a wide variety of functional categories. There were 11 interactors involved in metabolism, 9 interactors involved in transport, 8 interactors with unknown function, 4 interactors involved in trafficking and secretion, 3 interactors involved in protein folding, 2 interactors involved in stress response, and 1 interactor in each of the following categories: cell wall assembly, cytoskeleton maintenance, nuclear function, protein degradation, protein modification and protein synthesis. Follow up experiments also showed that Pdr15p and Pdr18p play an important role in zinc homeostasis because deletion of these ABC transporters results in sensitivity to zinc shock.

RNase footprinting and nitrocellulose filter-binding assays were previously used to map one major and two minor binding sites for the cell protein eEF1A on the 3’(+) stem loop (SL) RNA of West Nile virus (WNV) (2). Base substitutions in the major eEF1A binding site or adjacent areas of the 3’(+) SL were engineered into a WNV infectious clone. Mutations that decreased, as well as ones that increased, eEF1A binding in in vitro assays had a negative affect on viral growth. None of these mutations affected the efficiency of translation of the viral polyprotein from the genomic RNA, but all of the mutations that decreased in vitro eEF1A binding to the 3’ SL RNA also decreased viral minus-strand RNA synthesis in transfected cells. Also, mutations that increased the efficiency of eEF1A binding to the 3’ SL RNA increased minus-strand RNA synthesis in transfected cells, which resulted in decreased synthesis of genomic RNA. These results strongly suggest that the interaction between eEF1A and the WNV 3’ SL facilitates viral minus-strand initiation. eEF1A colocalized with viral replication complexes (RC) in infected cells and antibody to eEF1A coimmunoprecipitated viral RC proteins, suggesting that eEF1A facilitates an interaction between the 3’ end of the genome and the RC. eEF1A bound with similar efficiency to the 3’ terminal SL RNAs of four divergent flaviviruses, including a tick-borne flavivirus, and colocalized with dengue RC in infected cells. These results suggest that eEF1A plays a similar role in the RNA replication of all flaviviruses.

RNase footprinting and nitrocellulose filter-binding assays were previously used to map one major and two minor binding sites for the cell protein eEF1A on the 3’(+) stem loop (SL) RNA of West Nile virus (WNV) (2). Base substitutions in the major eEF1A binding site or adjacent areas of the 3’(+) SL were engineered into a WNV infectious clone. Mutations that decreased, as well as ones that increased, eEF1A binding in in vitro assays had a negative affect on viral growth. None of these mutations affected the efficiency of translation of the viral polyprotein from the genomic RNA, but all of the mutations that decreased in vitro eEF1A binding to the 3’ SL RNA also decreased viral minus-strand RNA synthesis in transfected cells. Also, mutations that increased the efficiency of eEF1A binding to the 3’ SL RNA increased minus-strand RNA synthesis in transfected cells, which resulted in decreased synthesis of genomic RNA. These results strongly suggest that the interaction between eEF1A and the WNV 3’ SL facilitates viral minus-strand initiation. eEF1A colocalized with viral replication complexes (RC) in infected cells and antibody to eEF1A coimmunoprecipitated viral RC proteins, suggesting that eEF1A facilitates an interaction between the 3’ end of the genome and the RC. eEF1A bound with similar efficiency to the 3’ terminal SL RNAs of four divergent flaviviruses, including a tick-borne flavivirus, and colocalized with dengue RC in infected cells. These results suggest that eEF1A plays a similar role in the RNA replication of all flaviviruses.

Immediately following a gene duplication event, if both gene copies are to be fixed into a species’ genome there is a period of enhanced selection acting on either one or both duplicates (paralogs) that results in some extent of functional divergence. However, as redundancy among extant duplicates is thought to confer genomic robustness, a consequent question is: how much functional overlap exists between duplicates that are retained over long spans of evolutionary time? To examine this issue I determined the extent of shared protein interactions and protein complex membership for paralogous gene pairs resulting from an ancient Whole Genome Duplication (WGD) event in yeast, finding retained functional overlap to be substantial among this group. Surprisingly however, I found paralogs existing within the same complex tended to maintain greater disparities in expression, suggesting the existence of previously proposed “transcriptional back-up” mechanisms. To test both for existence of such mechanisms and for any phenotypic manifestation of their shared functional overlap I surveyed for the presence of aggravating genetic interactions between 399 WGD-resultant paralog pairs. While these paralogs exhibited a high frequency (~30%) of epistasis, observed genetic interactions were not predictable based on protein interaction overlap. Further, exposure to a limited number of stressors confirmed that additional instances of epistasis were only observable under alternate conditions. As only a small number of stress conditions were tested, the high frequency of genetic interactions reported appears to be a minimum estimate of the true extent of epistasis among WGD paralogs, potentially explaining the lack of overlap with protein interaction data. As it is impossible to survey an infinite condition space, Synthetic Genetic Array (SGA) screening of yeast strains carrying double-deletions of paralog pairs was used to assess functional redundancy among a group of the remaining non-epistatic paralog pairs. The resulting interactions demonstrated functional relationships in non-epistatic paralogs only obvious upon ablation of both duplicates, suggesting that these interactions had initially been masked through redundant function. These findings ultimately suggest an advantage to retained functional overlap among whole genome duplicates that is capable of being stably maintained through millions of years of evolutionary time.

<p> Bacterial cell division is an essential process, which is initiated by forming the Z-ring as a cytoskeletal scaffold at the midcell site, followed by the recruitment of a series of divisome proteins. In <i>Escherichia coli</i> (Ec), at least 15 divisome proteins (FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsB, FtsL, FtsI, FtsW, FtsN, FtsE, FtsX, ZapA, AmiC, EnvC) have been implicated in this process. The components of the cell division machinery proteins in <i>Neisseria gonorrhoeae</i> (Ng) differs from <i>E. coli. N. gonorrhoeae</i> possesses FtsA, but lacks FtsB. ZipA and FtsL in <i>N. gonorrhoeae</i> have low identity to ZipA and FtsL from <i>E. coli</i>. Our laboratory has studied the central division protein FtsZ in <i>N. gonorrhoeae</i>. Thus, my research investigated the role of <i>N. gonorrhoeae</i> FtsA in cell division and investigated the interactions between divisome proteins from <i>N. gonorrhoeae</i> to understand divisome assembly.</p> <p>This study determined the association of FtsA_Ng with FtsZ</sub>Ng and other divisome proteins in <i>N. gonorrhoeae</i> and identified the functional domains of FtsA<sun>Ng</sub> involved in these interactions using a bacterial two-hybrid (B2H) assay. FtsA_Ng interacted with FtsZ_Ng, FtsK_Ng, FtsW_Ng, FtsQ_Ng, and FtsN_Ng. Self-interactions of FtsA_Ng and FtsZ_Ng were also detected. FtsI_Ng, FtsE_Ng and FtsX_Ng did not interact with FtsA_Ng. The 2A₁, 2A₂ and 2B domains of FtsA_Ng were sufficient to interact with FtsZ_Ng independently. Domain 2A₁ interacted with FtsK_Ng and FtsN_Ng. Domain 2B of FtsA_Ng interacted with FtsK_Ng, FtsQ_Ng, and FtsN_Ng. Domain 2A₂ of FtsA_Ng interacted with FtsQ_Ng, FtsW_Ng, and FtsN_Ng. These data suggest that FtsA in <i>N. gonorrhoeae</i> plays a key role in interactions with FtsZ and other divisome proteins.</p> <p>The potential interactions between divisome proteins in <i>N. gonorrhoeae</i> were examined using B2H assays. The comparisons between the <i>N. gonorrhoeae</i> divisome protein interaction network and those of <i>E. coli</i> and <i>S. pneumoniae</i> indicates that the divisome protein interactome of <i>N. gonorrhoeae</i> is more similar to that of <i>S. pneumoniae</i> and differs from that of <i>E. coli</i>. The comparisons revealed that compared to the interactions in <i>E. coli</i> and <i>S. pneumoniae</i>, more interactions between divisome proteins upstream of FtsA_Ng (including FtsA_Ng) and downstream of FtsA_Ng were observed in <i>N. gonorrhoeae</i> while fewer interactions between divisome proteins downstream of FtsA_Ng were observed in <i>N. gonorrhoeae</i>. Possible reasons for this include the inability of ZipA_Ng to interact with other divisome proteins and the absence of FtsL and FtsB in <i>N. gonorrhoeae</i>, resulting in the lack of an FtsQ-FtsB-FtsL complex in <i>N. gonorrhoeae</i>. These results indicate a possibly different divisome assembly in <i>N. gonorrhoeae</i> from that proposed models for <i>E. coli</i>.</p> A model for FtsA_Ng structure was predicted based on structural homology modeling with the resolved crystal structure of <i>Thermotoga maritima</i> FtsA. Four domains on the molecule were identified, designated 1A, 1C, 2B and 2A (including 2A₁ and 2A₂). Domains 2A and 2B of FtsA were highly conserved based on multi-sequence alignments of FtsAs from 30 bacteria. FtsA_Ng located to the division site in <i>N. gonorrhoeae</i> cells and the ratio of FtsA to FtsZ ranged from 1:24 to 1: 33 in three <i>N. gonorrhoeae</i> strains, which gave a lower cellular concentration of FtsA compared to other organisms.</p> <p>I also determined that overexpression of FtsA_Ng in <i>E. coli</i> led to cell filamentous in rod-shaped <i>E. coli</i> and cell enlargement and aggregation in mutant, round <i>E. coli</i>. FtsA_Ng failed to complement an <i>ftsA</i>_Ec-deletion <i>E. coli</i> strain although the overexperssion of FtsA_Ng disrupted <i>E. coli</i> cell division. In addition, overexpression of FtsA_Ng only affected cell division in some cells and its localization in <i>E. coli</i> was independent of interaction with <i>E. coli</i> FtsA or FtsZ. These results indicate that FtsA_Ng exhibits a species-specific functionality and <i>E. coli</i> is not a suitable model for studying FtsA_Ng functionality.</p> <p>This is the first study to characterize FtsA from <i>N. gonorrhoeae</i> in cell division. I identified novel functional domains of FtsA_Ng involved in interactions with other divisome proteins. The <i>N. gonorrhoeae</i> divisome protein interaction network determined by B2H assays provides insight into divisome assembly in <i>N. gonorrhoeae</i></p>.

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.