A major challenge in biology is to understand and predict the effect of mutations on protein structure, stability and function. Chapter 1 provides a general introduction on protein sequence-structure relationships and use of the CcdAB toxin-antitoxin system as a model to study molecular determinants of mutant phenotypes. In Chapter 2, we describe the use of saturation mutagenesis combined with deep sequencing to determine phenotypes for 1664 single-site mutants of the E. coli cytotoxin, CcdB. We examined multiple expression levels, effects of multiple chaperones and proteases and employed extensive in vitro characterization to understand how mutations affect these phenotypes. While general substitution preferences are known, eg polar residues preferred at exposed positions and non-polar ones at buried positions, we show that depth from the surface is important and that there are distinctly different energetic penalties for each specific polar, charged and aromatic amino acid introduced at buried positions. We also show that over-expression of ATP independent chaperones can rescue mutant phenotypes. Other studies have primarily looked at effects of ATP dependent chaperone expression on phenotype, where it is not possible to say whether mutational effects on folding kinetics or thermodynamic stability are the primary determinant of altered phenotypes, since there is energy input with these chaperones. The data suggest that mutational effects on folding rather than stability determine the in vivo phenotype of CcdB mutants. This has important implications for efforts to predict phenotypic effects of mutations and in protein design. While looking at the mutational landscape of a given gene from an evolutionary perspective, it is important to establish the genotype-phenotype relationships under physiologically relevant conditions. At the molecular level, the relationship between gene sequence and fitness has implications for understanding both evolutionary processes and functional constraints on the encoded proteins. Chapter 3 describes a methodology to test the fitness of individual CcdB mutants in E.coli over several generations by monitoring the rate of plasmid loss. We also propose a methodology for high throughput analysis of a pool of CcdB mutants using deep sequencing to quantitate the relative population of each mutant in a population of E.coli cells, grown for several generations and build the fitness landscape. While the F-plasmid based CcdAB system is known to be involved in plasmid maintenance through post-segregational killing, recent identification of ccdAB homologs on the chromosome, including in pathogenic strains of E.coli and other bacteria, has led to speculations on their functional role on the chromosome. In Chapter 4, we show that both the native ccd operon of the E.coli O157 strain as well as the ccd operon from the F- plasmid when inserted on the E.coli chromosome lead to protection from cell death under multiple antibiotic stress conditions through formation of persisters. Both the ccdF and ccdO157 operons may share common mechanisms for activation under stress conditions and also display weak cross activation. The chromosomal toxin shows weaker activity as compared to the plasmidic counterpart and is therefore less efficient in causing cell death. This has important implications in generation of potential therapeutics that target these TA systems. Chapter 5 describes the use of site-saturation mutagenesis coupled with deep sequencing to infer mutational sensitivity for the intrinsically disordered antitoxin, CcdA. The data allows us to make comparisons between overall as well as residue specific mutational sensitivity patterns with that of globular proteins, like CcdB (described in Chapter 2) and study toxin- antitoxin interaction and regulation through saturation suppressor mutagenesis. Interestingly, we found several examples of synonymous point mutations in CcdA that lead to loss of its activity. In Chapter 6 we attempt to explore the molecular bases for some of these synonymous mutations. In most cases the mutated codon has a similar overall codon preference to the WT one. Initial findings suggest a change in mRNA structure leading to change in CcdB: CcdA ratio, thereby causing cell death. These observations have important implications, because TA systems are ubiquitous, highly regulated and are known to be involved in multiple functions including drug tolerance. However a role for RNA structure in their regulation has not been shown previously. Appendix–I lists the mutational sensitivity scores for the CcdB mutants. Phenotypes for CcdA mutants obtained through deep sequencing have been tabulated in Appendix-II. Overall, we provide extensive datasets for mutational sensitivities of a globular (CcdB) and an intrinsically disordered protein (CcdA). Exploration of the molecular determinants of these mutant phenotypes not only provides interesting insights into CcdAB operon function but is also useful in understanding various aspects of protein stability, folding and activity as well as regulation of gene expression in bacteria.

This diploma thesis occupies itself with the subject of near synonymy, concretely with adjectives. On the basis of corpus linguistic methods two pairs of near synonyms have been researched - verschieden/unterschiedlich and bedeutend/bedeutsam. The 15 primary collocators (according to the syntactic position of each adjective) have been examined using the InterCorp parallel corpus methods in order to find out the most frequent Czech equivalence. Keywords: lexical-semantic relations, near synonymy, lexicography, corpora, cooccurrence analysis, Self Organizing Maps, CCDB

Genomic and proteomic investigations have yielded, and continue to produce, a large amount of information about genes and their protein products. In contrast, the evidence bearing on physiological roles of specific proteins is much more scarce. To address the functional part of biological inquiry, one would like to perturb, at will and selectively, the function of any protein of interest in vivo and to analyze the resulting phenotypic effects, thereby probing the protein’s role in a cell. Ideally, a method for doing so should be applicable both to individual gene products and to a large collection of them. Gene knockouts, a powerful tool to study gene function, have limitations in the study of development when the early phenotypes are cell- or organismal- lethal. Conditional mutants are particularly useful for analysis of genes whose functions are essential for the organism’s viability. A conditional mutant retains the function of a gene under one set of conditions, called permissive, and shows an inactive phenotype under a different set of conditions, called nonpermissive; the latter must be still permissive for the wild type (wt) allele of a gene. Conditional mutants make possible the analysis of physiological changes that follow controlled inactivation of a gene or gene product and can be used to address the function of any gene. Temperature sensitive (ts) mutants are an important class of conditional mutants whose phenotype is similar to that of wt at lower (permissive) temperature, but show low or reduced level of activity above a certain temperature called restrictive temperature, while the wt gene shows a similar phenotype at both the temperatures. Ts mutants provide an extremely powerful tool to study gene expression in vivo and in cell culture. They provide a reversible mechanism to lower the level of a specific gene product simply by changing the temperature of growth of the organism. Ts mutants are typically generated by random mutagenesis; either by ultraviolet light, a chemical mutagen or by error-prone PCR followed by often laborious screening procedures. Therefore, they are cumbersome to make, especially in the case of organisms with long generation times. Keeping in view the importance of ts mutants in biology, Varadarajan et al. 1996, had developed an algorithm to predict ts mutants at predicted, buried sites of a globular protein from its amino acid sequence. Experimental tests of the algorithm were carried out on the CcdB toxin of Escherichia coli to further refine and improve the method (Chakshusmathi et al. 2004). Based on this result simple rules for the design of ts mutants were suggested. This thesis aims at validating and improving on these rules and to find out if ts mutants of a protein can also be generated by perturbing functionally important residues. In addition, it is currently unclear with what frequency ts mutants of a protein isolated in one organism will show a ts phenotype in a completely different organism. This thesis makes preliminary efforts to address this issue. The model system chosen to carry out these studies is a protein called Gal4, which is a yeast transcriptional activator. This protein is biologically relevant as it has been used for ectopic gene expression in diverse organisms including yeast, fruitflies, zebrafish, mice and frogs (Ornitz et al. 1991; Brand and Perrimon 1993; Rahner et al. 1996; Andrulis et al. 1998; Scheer and Camnos-Ortega 1999; Hartley et al. 2002). The introductory chapter (Chapter 1) discusses the importance of ts mutants and our understanding and progress in this field so far, relevant for the work reported in this thesis. Chapter 2 describes generation of ts mutants of Gal4 in yeast. Full length Gal4 (fGal4) is an 881-aa protein. To simplify the construction of ts Gal4, we have designed a functional truncated Gal4 (miniGal4 or mGal4) of 197 residues. Five residues (9, 10, 15, 18 and 23) of the Gal4 DNA binding domain, which are in close contact with the DNA, were randomized in mGal4. Based on average hydrophobicity and hydrophobic moment, 68, 69, 70, 71, and 80 are the only residues in the region 1-150 that are predicted to be buried at the 90% confidence level. Of these five sites, residues 68, 69 and 70 were chosen for mutagenesis. At these three sites, four stereochemically diverse substitutions (Lys, Ser, Ala and Trp) were made. In a separate set of experiments each predicted, buried residues were also individually randomized in both mini and in full length Gal4 (fGal4). In all cases, we have been successful in isolating ts mutants in more than one position. At both permissive and restrictive temperatures, the activity of the Gal4 ts mutants is substantially lower than the wt. However, at the restrictive temperature, the activity of the ts Gal4 is lowered below the threshold required for reporter gene expression. This view of how ts mutants function is quite different from the general notion that the ts and wt behave similarly at permissive temperatures. Chapter 3 deals with transferability of two of the ts constructs mutated at DNA binding residues (R15W and K23P) to Drosophila. Two fGal4 encoding DNA fragments carrying the mutations were cloned into P element vectors under control of Elav and GMR promoters and several transgenic Drosophila lines were generated. These were crossed to various UAS reporter lines and progeny were characterized for reporter gene expression as a function of temperature. We show that both of these yeast ts mutants also show a ts phenotype in Drosophila. We have compared our ts Gal4 system with a popularly used system (TARGET) (McGuire et al. 2003) used for conditional gene expression in Drosophila. Our ts Gal4 mutants appear to provide tighter control at the restrictive temperature and a more uniform and rapid induction of gene expression upon shifting from the restrictive to the permissive temperature than the TARGET system with the promoters and the reporters we have used. Although cold sensitive (cs) mutants are often more useful than ts mutants, for reasons currently unclear, cs mutants are much more difficult to isolate than ts mutants. In Chapter 4, we have attempted to convert the ts phenotypes observed with Gal4 mutants in Drosophila and CcdB mutants in E. coli (Chakshusmathi et al. 2004) to cs phenotypes by increasing the expression level of these mutant proteins selectively at higher temperature. Several ts mutants of CcdB have been previously reported (Chakshusmathi et al. 2004). For converting the ts phenotype observed by E. coli toxin CcdB mutants (Chakshusmathi et al. 2004) to a cs phenotype, the arabinose inducible plasmid pBAD24CcdB and its mutant derivatives were used. By inducing expression of the mutant protein at higher temperature with arabinose, while keeping the basal level of expression without arabinose at lower temperature, we have been able to show cold sensitive behavior by these CcdB ts mutants in E. coli. For producing a cs phenotype with Gal4 mutants in Drosophila, we have used a P element vector where the GMR element is placed in-between hsp70 binding sites. This driver results in enhanced expression of downstream genes at 30 relative to 18°C because of the presence of the hsp elements (Kramer and Staveley 2003). Ts mutants at DNA binding and buried residues of fGal4 were cloned into this vector and several transgenic lines for each construct were obtained. The Gal4 mutants at exposed DNA binding residues but not at buried residues show a cs phonotype when they were crossed to various UAS-reporters lines. The buried residue mutants are likely to be destabilized and their degradation pathway might differ in yeast and in Drosophila. Because of this, these mutants might not be showing the desired cs phenotype in Drosophila. Although mGal4 and fGal4 have very similar activities in yeast, it was necessary to examine if they also had identical activities in Drosophila. Determining their relative activities in Drosophila is the aim of Chapter 5. To this end, mGal4 was cloned into P element vectors under control of hsp70 or GMRhs promoters and transgenic flies were generated. The transgenic lines were crossed to various UAS-reporters and reporter gene activities in the progeny were characterized. Although mGal4 and fGal4 showed similar activity in yeast, in Drosophila for reasons that are currently unclear, mGal4 was considerably less active than fGal4. As some of the fGal4 mutants showed a cs phenotype under GMRhs driver as shown in the earlier chapter (Chapter 4), several ts mutants of mGal4 in yeast in buried and as well as at the DNA binding residues were transferred to Drosophila under hs and GMRhs promoter. The transgenic lines obtained were tested for cold sensitivity by crossing with various UAS-reporter lines. However, in all cases mutant mGal4 showed an inactive phenotype in Drosophila. We suggest that this is because the intrinsic activity of these mGal4 mutants is substantially weaker than wt mGal4 even at permissive temperature in yeast. The further lowering of activity in Drosophila pushes the activity below the threshold required for reporter gene expression resulting in an inactive phenotype. The concluding chapter (Chapter 6) summarizes the conclusions drawn from this entire study and provides insights into possible mechanisms responsible for ts and cs phenotypes. The mutant phenotypes of Gal4 in yeast and in Drosophila suggest that ts phenotypes appear to result from a threshold effect. Such mutations lower the activity and/or level of the protein relative to the wt at all temperatures. Since maximal stability temperatures are rarely in excess of room temperature, with an increase in temperature, the activity of an already marginally active mutant can fall below the threshold required for function resulting in a temperature sensitive phenotype. The strategies we used for producing ts mutants have several advantages over standard approaches of generating ts alleles by random mutagenesis. We anticipate that conclusions of this study would be useful for generation of ts mutants of other globular proteins in diverse organisms. We also show that exposed, functional residues involved in protein: ligand or protein: protein interactions appear to be attractive candidate sites for generating ts mutants that are transferable between organisms. In addition, the active site mutants of fGal4 in Drosophila, which show ts and cs phenotypes depending on the Drosophila promoter chosen for expression, can be used for conditional and reversible expression of a number of other genes using the Gal4-UAS system (Brand and Perrimon 1993).

The unique native structure is a basic requirement for normal functioning of most proteins. Many diseases stem from mutations in proteins that destabilize the protein structure thereby resulting in impairment or loss of function (Sunyaev et al. 2000). Therefore, it is important from both fundamental and applied points of view, to elucidate the sequence determinants of protein structure and function. With the advent of recombinant DNA techniques for modifying protein sequences, studies on the effect of amino acid replacements on protein structure and function have acquired momentum. It is well established from previous mutagenesis studies that buried residues in a protein are important determinants of protein structure or stability while surface residues are involved in protein function (Rennell et al. 1991; Terwilliger et al. 1994; Axe et al. 1998). Inspite of this, there is no universally accepted definition and probe to distinguish and identify buried residues from exposed residues. A part of this thesis aims to examine the feasibility of using scanning mutagenesis to distinguish between buried and exposed positions in the absence of three-dimensional structure and also to arrive at an experimental definition of the appropriate accessibility cut-off to distinguish between buried and exposed residues. Proline, being an unusual amino acid is usually exploited to determine sites in a protein important for protein stability (Sauer et al. 1992). This thesis also explores the use of proline scanning mutagenesis to make inferences about protein structure and stability. Temperature sensitive mutant proteins, which result from single amino acid substitutions, are particularly useful in elucidating the determinants of protein folding and stability (Grutter et al. 1987; Sturtevant et al. 1989). Temperature sensitive (ts) mutants are an important class of conditional mutants which are widely used to study gene function in vivo and in cell culture (Novick and Schekman 1979; Novick and Botstein 1985). They display a marked drop in the level or activity of the gene product when the gene is expressed above a certain temperature (restrictive temperature). Below this temperature (permissive temperature), the level or activity of the mutant is very similar to that of the wild type. Inspite of their widespread use, little is known about the molecular mechanisms responsible for generating a Ts phenotype. A part of this thesis discusses a set of sequence/structure-based strategies for the successful design and isolation of ts mutants of a globular protein, inferred from saturation mutagenesis of CcdB. The experimental system, CcdB (Controller of Cell Division or Death B protein), is a 101 residue, homodimeric protein encoded by F plasmid. The protein is an inhibitor of DNA gyrase and is a potent cytotoxin in E.coli (Bernard et al. 1993). Crystallographic structures of CcdB in the free and gyrase bound forms (Loris et al. 1999; Dao-Thi et al. 2005) are also available. Expression of the CcdB functional protein results in cell death, thus providing a rapid and easy assay for the protein (Chakshusmathi et al. 2004). This dissertation focuses on understanding the determinants of globular protein stability and temperature sensitivity using saturation mutagenesis of E.coli CcdB. Towards this objective, we attempted to replace each of the 101 residues of CcdB with 19 other amino acids using high throughput mutagenesis tools. A total of 1430 (~75%) of all possible single site mutants of the CcdB saturation mutagenesis library could be isolated. These mutants were characterized in terms of their activity at different expression levels. The correlation between the observed mutant phenotypes with residue burial, nature of substitution and expression level was examined. The introductory chapter (Chapter 1) describes the use of mutagenesis as a tool to understand the relationship between protein sequence, structure and function. It represents an overview of previous large scale mutagenesis studies from the literature. It also addresses the motivation behind this work and problems which we have attempted to address in these studies. Chapter 2 discusses mutagenesis based definitions and probes for residue burial in proteins as derived from alanine and charged scanning mutagenesis of CcdB. Every residue of the 101 amino acid E. coli toxin CcdB was substituted with Ala, Asp, Glu, Lys and Arg using site directed mutagenesis. The activity of each mutant in vivo was characterized as a function of CcdB transcriptional level. The mutation data suggest that an accessibility value of 5% is an appropriate cutoff for definition of buried residues. At all buried positions, introduction of Asp results in an inactive phenotype at all CcdB transcriptional levels. The average amount of destabilization upon substitution at buried positions decreases in the order Asp>Glu>Lys>Arg>Ala. Asp substitutions at buried sites in two other proteins, MBP and Thioredoxin were also shown to be severely destabilizing. Ala and Asp scanning mutagenesis, in combination with dose dependent expression phenotypes, was shown to yield important information on protein structure and activity. These results also suggest that such scanning mutagenesis data can be used to rank order sequence alignments and their corresponding homology models, as well as to distinguish between correct and incorrect structural alignments. When incorporated into a polypeptide chain, Proline (Pro) differs from all other naturally occurring amino acids in two important respects. The  dihedral angle of Pro is constrained to values close to –65o and Pro lacks an amide hydrogen. Chapter 3 describes a procedure to accurately predict the effects of proline introduction on protein stability. 77 of the 97 non-Pro amino acid residues in the model protein, CcdB, were individually mutated to proline and the in vivo activity of each mutant was characterized. A decision tree to classify the mutation as perturbing or non-perturbing was created by correlating stereochemical properties of mutants to activity data. The stereochemical properties, including main chain dihederal angle and main chain amide hydrogen bonds, were determined from 3D models of the mutant proteins built using MODELLER. The performance of the decision tree was assessed on 74 nsSNPs and 37 other proline substitutions from the literature. The overall accuracy of this algorithm was found to be 89% in case of CcdB, 71% in case of nsSNPs and 83% in case of other proline substitution data. Contrary to previous assertions, Proline scanning mutagenesis cannot be reliably used to make secondary structural assignments in proteins. The studies will be useful in annotating uncharacterized nsSNPs of disease-associated proteins and for protein engineering and design. Mutants of CcdB were also characterized in terms of their activity at two different temperatures (30oC and 37oC) to screen for temperature sensitive (ts) mutants. The isolation and structural analysis of Ts mutants of CcdB is dealt with in Chapter 4. Of the total 1430 single site mutants, 12% showed a ts phenotype and were mapped onto the crystal structure of the protein. Almost all the ts mutants could be interpreted in terms of the wild type, native structure. ts mutants were found at all buried sites and all active sites (except one). ts mutants were also obtained at sites in close proximity to active site residues where polar side-chains were involved in H-bonding interaction with active site residues. Several proline substitutions also displayed a ts phenotype. The effect of expression level on ts phenotype was also studied. 78% of the mutants that showed an inactive phenotype at the lowest expression level and an active phenotype at highest expression level, resulted in a ts phenotype at an intermediate expression level. The molecular determinant responsible for the ts phenotype of buried site ts mutant is suggested to be the thermodynamic destabilization of the protein which results in a reduced steady state in vivo level of soluble, functional protein relative to wild type. The active site ts mutants probably lower the specific activity of the protein and hence the total activity relative to wild type. However these effects might be less severe at lower temperature. Specific structure/function based mutagenesis strategies are suggested to design ts mutant of a protein. These studies will simplify the design of ts mutants for any globular protein and will have applications in diverse biological systems to study gene function in vivo. Chapter 5 represents the structural and sequence correlations of a CcdB saturation mutagenesis library which was obtained by replacing each of 101 amino acid residues with 19 other amino acids. Polar substitutions i.e. Asn, Gln, Ser, Thr and His were poorly tolerated at buried sites at lower expression levels. Aromatic substitutions and Gly were also not well tolerated at buried positions at lower expression levels. Trp was poorly tolerated at residues with accessibility <15%. However, most of the surface exposed residues with accessibility >40% (except functional ones) could tolerate all kinds of substitutions. Chapter 6 deals with the thermodynamic characterization of monomeric and dimeric forms of CcdB. The stability and aggregation state of CcdB have been characterized as a function of pH and temperature. Size exclusion chromatography revealed that the protein is a dimer at pH 7.0, but a monomer at pH 4.0. CD analysis and fluorescence spectroscopy showed that the monomer is well folded, and has similar tertiary structure to the dimer. Hence intersubunit interactions are not required for folding of individual subunits. The oligomeric status of CcdB at pH 7.0 at physiologically relevant low concentrations of protein, was characterized by labeling the protein with two different pairs of donor and acceptor fluorescent dyes (Acrylodan-Pyrene and IAF-IAEDANS) separately and carrying out fluorescence resonance energy transfer (FRET) measurements by mixing them together. CcdB exists in a dimeric state even at nanomolar concentrations, thus indicating that the dimeric form is likely to be the physiologically active form of CcdB. The stability of the dimeric form at pH 7.0 and the monomeric form at pH 4.0 was characterized by isothermal denaturant unfolding and calorimetry. The free energies of unfolding were found to be 9.2 kcal/mol (1 cal=4.184 J) and 21 kcal/mol at 298 K for the monomer and dimer respectively. The denaturant concentration at which one-half of the protein molecules are unfolded (Cm) for the dimer is dependent on protein concentration, whereas the Cm of the monomer is independent of protein concentration, as expected. Although thermal unfolding of the protein in aqueous solution is irreversible at neutral pH, it was found that thermal unfolding is reversible in the presence of GdnCl (guanidinium chloride). Differential scanning calorimetry in the presence of low concentrations of GdnCl in combination with isothermal denaturation melts as a function of temperature were used to derive the stability curve for the protein. The value of Cp (representing the change in excess heat capacity upon protein denaturation) is 2.8 ± 0.2 kcalmol-1K-1 for unfolding of dimeric CcdB, and only has a weak dependence on denaturant concentration. These studies advanced the understanding of protein folding of oligomeric proteins. The concluding section summarizes all the chapters in a nutshell and addresses the future directions provided by these investigations.

The work concerns the study of limited collocational paradigms in modern German. It is based on the corpus cooccurrence analysis and aims at the clearing up of the phenomenon of the so- called monocollocability. After the undertaken analyses and reflections there came a conclusion that corroborated the original point of view, namely, that the so-called monocollocability presents a multiplex phenomenon, where a metaphore of centre and periphery seems to be appropriate. In the analyses there occur semantically neutral word, inherently bound on certain collocated for formal reasons.

This paper deals with corpus linguistics. There are two applications under its scrutiny. Both of these applications are processing data from the corpus DeReKo via corpus-driven approach. It is a co-occurrence analysis and a Co-occurrence database. The aim of the work is to evaluate whether the results obtained by the co-occurrence analysis of the current scope of DeReKo are different from the results of the Co-occurrence database, which was created on a basis of a smaller scale corpus. In addition, this thesis offers illustrative examples of the use of both applications and the evaluation of their effectiveness, depending on the purpose of the research. The theoretical part of the thesis deals with the terminology of corpus linguistics and with the mentioned corpuses, which serve as a basis for the practical part of the thesis. The empirical part of the thesis consists of analyses of the randomly picked words (one from each word class) in both applications. The results confirm that the data obtained with Co-occurrence database and co-occurrence analysis are in many respects different and thus confirm the hypothesis that the corpus size plays a crucial role in the results. Both applications have their advantages and disadvantages. The paper offers a comprehensive overview and by doing so it...

The sequence of a polypeptide chain determines its structure which in turns determines its function. A protein is stabilized by multiple forces; hydrophobic interaction, electrostatic interactions and hydrogen bond formation between residues. While the above forces are non-covalent in nature the protein structure is also stabilized by disulfide bonds. Structural features such as naturally occurring cavities in proteins also affect its stability. Studying factors which affect a protein’s structural stability helps us understand complex sequence-structure-function relationships, the knowledge of which can be applied in areas such as protein engineering. The work presented in this thesis deals with various and diverse aspects of protein structure. Chapter 1 gives an overall introduction on the topics studied in this thesis. Chapter 2 focuses on a unique, non-regular, structural feature of proteins, viz. protein cavities. Cavities directly affect the packing density of the protein. It has been shown that large to small cavity creating mutations destabilize the protein with the extent of destabilization being proportional to the size of cavity created. On the other hand, small to large cavity filling mutations have been shown to increase protein stability. Tools which analyze protein cavities are thus important in studies pertaining to protein structure and stability. The chapter presents two methods which detect and calculate cavity volumes in proteins. The first method, DEPTH 2.0, focuses on accurate detection and volume calculation of cavities. The second method, ROBUSTCAVITIES, focuses on detection of biologically relevant cavities in proteins. We then study another aspect of protein structure – the disulfide bond. Disulfide bonds confer stability to the protein by decreasing the entropy of the unfolded state. Previous studies which attempted to engineer disulfides in proteins have shown mixed results. Previously, disulfide bonds in individual secondary structures were characterized. Analysis of disulfides in α-helices and antiparallel β-strands yielded important common features of such bonds. In Chapter 3 we present a review of these studies. We then use MODIP; a tool that identifies amino acid pairs which when mutated to cysteines will most likely form a disulfide bond, to analyze disulfide bonds in parallel β-strands. A direct way to analyze sequence-structure relationships is via mutating individual residues, evaluating the effect on stability and activity of the protein and inferring its effect on protein structure. Saturation mutagenesis libraries, where all possible mutations are made at every position in the protein contain a huge amount of information pertaining to the effect of mutations on structure. Making such libraries and screening them has been an extremely resource intensive process. We combine a fast inverse PCR based method to rapidly generate saturation mutagenesis libraries with the power of deep sequencing to derive phenotypes of individual mutants without any large scale screening. In Chapter 4 we present an Illumina data analysis pipeline which analyzes sequencing data from a saturation mutagenesis library, and derives individual mutant phenotypes with high confidence. In Chapter 5 we apply the insights derived from structure-function studies and apply it to the problem of protein engineering, specifically immunogen design. The Human Immunodeficiency Virus adopts various strategies to evade the host immune system. Being able to display the conserved epitopes which elicit a broadly neutralizing response is the first step towards an effective vaccine. Grafting such an epitope onto a foreign scaffold will mitigate some of the key HIV defenses. We develop a computational protocol which grafts the broadly neutralizing antibody b12 epitope on scaffolds selected from the PDB. This chapter also describes the only experimental work presented in this thesis viz. cloning, expressing and screening the epitope-scaffolds using Yeast Surface Display. Our epitope-scaffolds show modest but specific binding. In a bid to improve binding, we make random mutant libraries of the epitope-scaffolds and screen them for better binders using FACS. This work is on-going and we aim to purify our epitope-scaffolds, characterize them biophysically and eventually test their efficacy as immunogens.