Disulfides are the primary covalent interactions within a protein molecule that connect residues which are sequentially distant. Naturally occurring disulfides enhance the stability of the protein by destabilization of the unfolded state. Previous attempts to introduce disulfide bridges as a means to enhance protein stability have met with mixed results. Tools have been developed to predict potential sites for disulfide introduction. However, it must be noted that engineering disulfides is not a trivial task. The effect of the engineered disulfide on protein stability is difficult to predict. There have been few systematic studies carried out to study disulfides in the context of secondary structures. The work in this thesis is aimed at studying disulfides in two kinds of secondary structures- antiparallel β-sheets and helices. In particular, the focus in this thesis is on cross-strand disulfides in antiparallel β-sheets and intrahelical disulfides. The analysis of naturally occurring disulfides in these structural elements coupled with protein engineering studies in model proteins were used to understand the effects of introducing disulfides in helices and antiparallel β-sheets. Synopsis This thesis also includes studies carried out on molten globules of four periplasmic binding proteins of E.coli- Maltose binding protein (MBP), Leucine, isoleucine, valine binding protein (LIVBP), Leucine binding protein (LBP) and Ribose binding protein (RBP). Work carried out in the lab previously had shown that these molten globules can bind the ligands that the proteins do in their corresponding native states. The analysis of the thermodynamic data obtained for these molten globules by differential scanning calorimetry (DSC) studies and isothermal titration calorimetry (ITC) to characterize stability and ligand binding respectively are described in this thesis. To further study the structural features of molten globules by fluorescence resonance energy transfer (FRET), double cysteine mutants of MBP were constructed and characterized. The rationale behind the construction of these mutants and their characterization is reported. Chapter 1 gives an introduction to disulfides in proteins. Previous attempts at cataloguing and characterizing naturally occurring disulfides are described. An overview of studies carried out to determine the effects of removal of naturally occurring disulfides in proteins and the effect of engineered disulfides in different proteins is given. The various tools developed to predict potential disulfide sites are described. Chapter 1 also briefly discusses various aspects of molten globules and FRET. Chapters 2 and 3 involve studies with cross-strand disulfides occurring in antiparallel β-sheets. A detailed analysis on various stereochemical aspects of naturally occurring cross-strand disulfides is described in Chapter 2. The reasons for these disulfides to almost exclusively occur at non-hydrogen-bonded registered pairs have been explored with conformational analysis, modeling studies and energy calculations. In Chapter 3, the effect of engineering cross-strand disulfides in four model proteins- LBP, LIVBP, MBP and Top7 are described. The ease of formation of the introduced disulfides and their effects on protein stability are described. The proteins with engineered cross-strand disulfides at exposed positions were also examined for redox activity. Our studies have shown that in antiparallel strands, engineered disulfides at exposed NHB registered pairs provide a robust means of increasing protein stability. In Chapters 4 and 5, studies about intrahelical disulfides are described. In Chapter 4, the various conformational aspects of intrahelical disulfides occurring naturally are studied. Analysis of structures of proteins in conjunction with modeling studies show that all naturally occurring intrahelical residues bridge cysteines occurring between the N-Cap and 3rd residue of helices. To further explore conformational requirements for intrahelical disulfides, Cys pairs were introduced at N-terminal and interior of helices in a E.coli thioredoxin mutant lacking its active site disulfide. The ease of formation of the engineered disulfides, and their effects on protein stability were studied. The redox activity of the engineered disulfides was also examined. The studies demonstrated that intrahelical disulfides can only occur at the N-terminus of an α-helix and that the N-terminal CYS residue must adopt a non-helical backbone conformation. Although none of the engineered intrahelical disulfides increased the stability of the protein, they conferred mild redox activity. In Chapter 5, the ability of an engineered CXXC motif to bind Zn(II) is also explored. The effect of Zn(II) on the stability of the reduced and oxidized states of an engineered protein with a N-terminal intrahelical CXXC was ascertained. I have also shown that iminodiacetate (IDA) and nitrilotriacetate (NTA) resins charged with zinc can bind the protein CGPC 95-98 in reduced state. These Synopsis preliminary experiments on metal binding show that this property of CXXC motif could be exploited to develop a protein purification method. In Chapter 6, thermodynamic characterization of molten globules of four periplasmic binding proteins (LBP, LIVBP, MBP and RBP) is described. Studies had been previously carried out in the lab to characterize the stability and ligand binding of these molten globules. All four molten globules were found to bind their corresponding ligands without conversion to the native state. In Chapter 6, the estimation of ΔCp of unfolding and ligand binding from the DSC and ITC data is described. The binding of molten globules to their ligands and the ability to undego cooperative thermal unfolding indicated the presence of native protein-like tertiary contacts. To study the molten globule structure, we decided to construct double cysteine mutants of MBP for FRET studies. We decided to employ a strategy for differential labeling of the two cysteines with two different fluorophores based on the conformational differences between MBP in the ligand bound and free forms. Seven double cysteine mutants of MBP were made. The rationale behind the construction of these mutants and their preliminary characterization is described in the appendix to Chapter 6. The optimization of the differential labeling procedure of the MBP double mutants needs to be fine-tuned before further studies through FRET. The work described in this thesis has resulted in the following publications: 1.Prajapati RS, Indu S, Varadarajan R. Identification and thermodynamic characterization of molten globule states of periplasmic binding proteins. Biochemistry. 2007 (46):10339-52. 1 Indu S, Kumar ST, Thakurela S, Gupta M, Bhaskara RM, Ramakrishnan C, Varadarajan R. Disulfide conformation and design at helix N-termini. Proteins.2010 (78):1228-42. 2 Indu S, Kochat V, Thakurela S, Ramakrishnan C, Varadarajan R. Conformational analysis and design of cross-strand disulfides in antiparallel β-sheets. (Manuscript submitted)