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)

Parmi les approches employées pour caractériser l’état sanitaire des populations antérieures, l’utilisation de l’asymétrie fluctuante (AF) pour rendre compte de la stabilité de développement (DS) du squelette est rare. Après plus d’un demi-siècle de recherches, la DS reste assez méconnue chez l’Homme. Aujourd’hui, les techniques de morphométrie géométrique permettent de quantifier plus finement les variations en analysant de nouveaux aspects morphologiques en 3D.Les objectifs de ce travail étaient:1-Déterminer si l’analyse morpho-géométrique de l’AF chez l’Homme permet de distinguer deux populations.2-Établir si cette distinction peut être attribuée à une différence d’état sanitaire.Les données de deux populations caractérisées par des contextes sanitaires distincts ont été confrontées. Deux échantillons de 70 paires de fémurs adultes d'âge et de sexe comparables ont été sélectionnés. Vingt-sept points-repères ont été positionnés sur chaque surface osseuse reconstruite en 3D. L’AF a été analysée en termes d’amplitude et de localisation sur le fémur.Finalement, cette analyse morpho-géométrique a permis de distinguer deux populations au contextes sanitaires distincts. La population la moins favorisée a exprimé un degré d’AF plus élevé. Considérant l’impact des facteurs potentiels de variation, le stress sanitaire explique le mieux les perturbations de la DS. Au vu de la localisation de l’AF sur le fémur, le facteur biomécanique semble aussi jouer un rôle relatif. Chez l’Homme, la localisation de l’asymétrie fluctuante peut donc être dirigée en partie par les contraintes biomécaniques, cependant son amplitude est principalement influencée par le stress sanitaire. / Among the approaches used to characterize the health status in past populations, the use of fluctuating asymmetry (FA) to evaluate developmental stability (DS) in bone is rare. After more than half a century of research, DS remains relatively unknown in humans. Today, geometric morphometrics techniques offer new perspectives. It is now possible to quantify variations more precisely and approach them by analyzing a new morphological element: shape.The aim of this work was twofold:1-Determine whether the geometric morphometrics analysis of FA in humans can distinguish two populations.2-Determine whether this distinction can be attributed to a difference in health status.The data of two populations of known and distinct health statuses and environmental contexts were compared. Two samples of 70 pairs of adult femurs of comparable age and sex were selected. Two sets of 27 landmarks were placed on the 3D isosurfaces of each femur. FA was analyzed and characterized in terms of range of magnitude and pattern of expression in the femur.Finally, this geometric morphometrics analysis allowed to distinguish two populations that have developed under distinct environmental conditions. The population that experienced higher levels of stress expressed a higher degree of FA. After considering the impact of potential factors of variation, the health stress seems to best explain disturbances of DS. However, given its pattern of expression on the femur, the biomechanical factor seems to play also a relative role. Therefore, in humans, the pattern of expression of FA is directed in part by biomechanical constraints but its magnitude remains mostly influenced by health stress.

Bacterial protein WrbA from E. coli is the founding member of a new family of FMN-dependent NAD(P)H oxidoreductases, forming a functional and structural bridge between bacterial flavodoxin and certain mammalian NAD(P)H:quinone oxidoreductase. For these reasons, protein WrbA is recently intensively studied using various analytical and computing methods. Protein WrbA participates in the protection of cells against oxidative stress, but precise function of the protein WrbA in vivo is still unknown. Protein WrbA forms multimers in solutions. In μM concentrations and at low temperature (4 řC) the protein is in the form of a dimer, with increasing temperature becomes tetrameric. Available three-dimensional crystal structure contains the information about the tetrameric form of the protein, the dimeric form has not been structurally characterized. This thesis was focused on the study of the dynamic behavior of protein WrbA in solution using methods of hydrogen-deuterium exchange and chemical cross-linking followed by mass spectrometric analysis with high resolution (FT-ICR). Behavior of the protein was monitored according to the presence of cofactor FMN. Effect of temperature and protein concentration was also studied. Hydrogen-deuterium exchange provided information about solvent accessibility and...

Molecular recognition, defined as the specific interactions between two or more molecules, is at the center of many biological processes including catalysis, signal transduction, gene regulation and allostery. Allosteric regulation is the modification of function caused by an intermolecular interaction. Allosteric proteins modify their activity in response to a biological signal that is often transmitted through the interaction with a small effector molecule. Therefore, determination of the origins of intermolecular interactions involved in molecular recognition and allostery are essential for understanding biological processes. Classically, molecular recognition and allosteric regulation have been associated to structural changes of the system. NMR spectroscopic methods have indicated that changes in protein dynamics may also contribute to molecular recognition and allostery. This thesis is an investigation of the contributions of both structure and dynamics in molecular binding phenomena. In chapter I, I describe molecular recognition, allostery and examples of allostery and cooperativity. Then I discuss the contribution of protein dynamics to function with a special focus on allosteric regulation. Lastly I introduce the hemoglobin homodimer, HbI of Scapharca inaequivalvis and the mRNA binding protein TIS11d. Chapter II is the primary focus of this thesis on the contribution of protein dynamics to allostery in the dimeric hemoglobin of scapharca inaequivalvis, HbI. Thereafter I concentrate on the mechanism of adenine recognition of the Tristetraprolin-like (TTP) protein TIS11d; this study is detailed in Chapter III. In Chapter IV I discuss broader impacts and future directions of my research. This thesis presents an example of the use of protein NMR spectroscopy to probe ligand binding. The studies presented in this thesis emphasize the importance of dynamics in understanding protein function. Measurements of protein motions will be an element of future studies to understand protein function in health and disease.

MHC II proteins are heterodimeric glycoproteins that form complexes with antigenic peptides in order to elicit a CD4+ adaptive immune response. Even though there have been numerous MHC II-peptide crystal structures solved, there is little insight into the dynamic process of peptide loading. Through biochemical and biophysical studies, it has been shown that MHC II adopt multiple conformations throughout the peptide loading process. At least one of these conformations is stabilized by the MHC II-like homologue, HLA-DM. The main focus of this thesis is to elucidate alternate conformers of MHC II in an effort to better understand the structural features that enable HLA-DM catalyzed peptide loading. In this thesis, two altered conformations of HLA-DR were investigated, one modeled in the absence of peptide using molecular dynamics, and one stabilized by the mutation αF54C. The model for the peptide-free form of HLA-DR1 was derived from a molecular dynamics simulation. In this model, part of the alpha-subunit extended-strand region proximal to the peptide binding groove is folded into the peptide-binding groove such that the architecture of the critical peptide binding pocket, P1, as well as the invariant hydrogen bonding network were maintained. Biochemical studies aimed at validating the predicted structural changes were consistent with the model generated from the simulations. Next, structural studies were carried out on an MHC II mutant, αF54C, which was shown to have unique peptide binding characteristics as well as enhanced susceptibility to HLA-DM. Although this mutation did not affect the affinity for peptide, there was a striking increase in the rate of intrinsic peptide release. Both αF54C and αF54A were over 100-fold more susceptible to HLADM catalyzed peptide release than wild type as well as other mutants introduced along the peptide binding groove. In addition, mutation of the αF54 position results in a higher affinity for HLA-DM, which, unlike wild type, is detectable by surface plasmon resonance. Crystallographic studies resulted in a 2.3 Å resolution structure for the αF54C-Clip complex. There were two molecules in the asymmetric unit, one of which had no obvious deviations from other MHC II-pep complexes and one which had a conformational change as a result of a crystal contact on the αF51 residue, a residue which has been shown to be involved in the HLA-DM/HLA-DR binding interface. The crystal structure of wild type HLA-DR1- Clip was also solved, but did not have the altered conformation even though there was a similar crystal contact at the αF51. These data suggest the altered conformation seen in the mutant structure, results from increased lability in the extended stand region due to the αF54C mutation. As a result of this work, we have developed a new mechanistic model for how structural features of MHC II influence DM mediated peptide release.

A hierarchy of different chromosome conformations plays a role in many biological systems. These conformations contribute to the regulation of gene expression, cellular development, chromosome transmission, and defects can lead to human disease. The highest functional level of this hierarchy is the partitioning of the genome into compartments of active and inactive chromatin domains (1’s -10’s Mb). These compartments are further partitioned into Topologically Associating Domains (TADs) that spatially cluster co-regulated genes (100’s kb – 1’s Mb). The final level that has been observed is long range loops formed between regulatory elements and promoters (10’s kb – 100’s Mb). At all of these levels, mechanisms that establish these conformations remain poorly understood. To gain new insights into processes that determine chromosome folding I used the mating type switching system in budding yeast to study the chromosome conformation at length scales analogous to looping interaction. I specifically examined the role in chromosome conformation in the mating type switching system. Budding yeast cells can have two sexes: MATa and MATα. The mating types are determined by allele-specific expression of the MAT locus on chromosome III. The MATa allele encodes for transcription factors responsible for the MATa mating type and the MATα allele encodes transcription factors responsible for the MATα mating type. Yeast cells can switch their mating type by a process that repairs a break at MAT using one of two silent loci, HML or HMR, as a donor to convert the allele at the MAT locus. When MATa cells switch they prefer to use HML, which contains the MATα allele, located at the end of the left arm. MATα cells prefer to use HMR, which contains the MATa allele, located on the end of the right arm of chromosome III. The sequences of the HM loci are not important for donor preference. Instead the cell chooses the donor on the left arm in MATa cells and chooses the donor on the right arm in MATα cells. This lack of sequence specificity has led to the hypothesis that the conformation of the chromosome may play a role in donor preference. I found that the conformation of chromosome III is, indeed, different between the two mating types. In MATa cells the chromosomes displays a more crumpled conformation in which the left arm of the chromosome interacts with a large region of the right arm which includes the centromere and the MAT locus. In MATα cells, on the other hand, the left arm of the chromosomes displays a more extend conformation. I found that the Recombination Enhancer (RE), which enhances recombination along the left arm of the chromosome in MATa cells, is responsible for these mating type-specific conformations. Deleting the RE affects the conformation of the chromosomes in both MATa and MATα cells. The left portion of the RE, which is essential for donor preference during the switching reaction in MATa cells, does not contribute to the conformation in MATa. This region does have a minor effect on the conformation in MATα cells. However, I found that the right portion of the RE is responsible for the conformation of chromosome III in both mating types prior to initiation of switching. This work demonstrates that chromosome conformation is determined by specific cis regulatory elements that drive cell-type specific chromosome conformation.

Indiana University-Purdue University Indianapolis (IUPUI) / Intrinsically disordered proteins (IDPs) are abundant in nature, being more prevalent in the proteomes of eukaryotes than those of bacteria or archaea. As introduced in Chapter I, these proteins, or portions of these proteins, lack stable equilibrium structures and instead have dynamic conformations that vary over time and population. Despite the lack of preformed structure, IDPs carry out many and varied molecular functions and participate in vital biological pathways. In particular, IDPs play important roles in cellular signaling that is, in part, enabled by the ability of IDPs to mediate molecular recognition. In Chapter II, the role of intrinsic disorder in molecular recognition is examined through two example IDPs: p53 and 14-3-3. The p53 protein uses intrinsically disordered regions at its N- and C-termini to interact with a large number of partners, often using the same residues. The 14-3-3 protein is a structured domain that uses the same binding site to recognize multiple intrinsically disordered partners. Examination of the structural details of these interactions highlights the importance of intrinsic disorder and induced fit in molecular recognition. More generally, many intrinsically disordered regions that mediate interactions share similar features that are identifiable from protein sequence. Chapter IV reviews several models of IDP mediated protein-protein interactions that use completely different parameterizations. Each model has its relative strengths in identifying novel interaction regions, and all suggest that IDP mediated interactions are common in nature. In addition to the biologic importance of IDPs, they are also practically important in the structural study of proteins. The presence of intrinsic disordered regions can inhibit crystallization and solution NMR studies of otherwise well-structured proteins. This problem is compounded in the context of high throughput structure determination. In Chapter III, the effect of IDPs on structure determination by X-ray crystallography is examined. It is found that protein crystals are intolerant of intrinsic disorder by examining existing crystal structures from the PDB. A retrospective analysis of Protein Structure Initiative data indicates that prediction of intrinsic disorder may be useful in the prioritization and improvement of targets for structure determination.

This diploma thesis deals with the conformation and molecular organization of humic acids in aqueous solutions. Humic substances have heterogeneous and polydisperse nature, therefore their secondary chemical structure has not yet been defined properly, neither has their conformational arrangement. The conformation of humic substances in the solutions is mainly stabilized by weak disperse forces, such as Van der Waals, -, CH- interactions and hydrogen bonds. Humic substances in the solutions tend to form aggregates that are held together mostly by the intermolecular hydrophobic interactions. Concentration series of humic acids were prepared in three different mediums of constant ionic strength: NaOH and NaCl (prepared either by neutralization NaOH by HCl or direct dilution by solution of NaCl). Several analytical methods have been used to determine conformation and molecular organization of humic acids: potentiometric determination of pH, direct conductometry, ultraviolet and visible spectroscopy, density measurement, dynamic light scattering, laser Doppler velocimetry and high resolution ultrasound spectrometry. It was confirmed that the conformation and molecular organization of humic acids in aqueous solutions depend not only on pH of medium, but they also depend on chemical nature and concentration with the same pH. Results showed that hydrodynamic diameter of particles is significantly increasing in NaCl medium prepared by neutralization NaOH by HCl, particularly at low concentrations, which corresponds to higher values of zeta potential obtained. Concentration dependencies of ultrasonic velocity and compressibility also indicate the changes in conformation and molecular organization corresponding with results from other methods used.

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The prevailing housing shortage and the developed considerations of environmental issues are todaytwo current topics. Bollnäs county is an example of a county that works to maintain and actualize itsenvironmental goals, especially in new constructions. Passive houses are a way to buildenvironmental friendly and it is a concept that is becoming more and more common. The concept isabout maximizing heat gains and minimizing heat losses. This is achieved through an air-tightenvelope, certified building components, exhaust and supply air ventilation where heat is regainedfrom the extract air and buildings with low U-values. This project presents a change of an existingdetailed development plan from general purpose to housing. The aim of the project is that thehouse, with a restaurant on the bottom floor, must meet the requirements for passive houses andharmonize with the surrounding environment.The project initiates with a literature study followed by an analytical study with the aim to gatherknowledge about both passive houses as well as the surrounding area of the property. Conformationand calculation are two essential parts of the project. The shape of the house, the design and wherethe house is located in the property are adjusted to both the requirements of passive houses and thecultural history of Bollnäs.The calculations performed in PHPP indicate that the requirements for passive houses are met on thebasis of the conditions in this project. Drawings such as site plan, general plans, facades, sections anddetails are presented together with illustrations of the finished house.The main problem regarding the projection of passive houses lies in the absence of its actualmeaning. The concept is about compensations and that every house, regardless of building systemand conformation, has the possibility to be certified as a passive house.