Master of Science (MSc)

Two computer simulation studies were performed; one to help understand the structure-function relationships of prolyl peptides (Part I) and the other to help predict more efficient pharmaceutical drug delivery by molecular modification of small peptides (Part II). In Part I, the free energy perturbation (FEP) method, using AMBER, was utilized to calculate the Gibbs free energy difference between <i>cis</i> and <i>trans</i> conformers of Ace-Tyr-Pro- NMe and Ace-Asn-Pro-NMe, from which the ratio of <i>cis</i> to <i>trans</i> conformers was obtained. Our simulation generated much lower <i>%cis</i> for both peptides as compared with experimental values and possible problems in our computational schemes are presented. However, our results were encouraging in that they predicted preference of <i>trans</i> conformers for both peptides and higher <i>%cis</i> for Ace-Tyr-Pro-NMe, compared to Ace-Asn-Pro-NMe, which agrees with experimental results. Part II applied semi empirical (AMS0L) and microscopic simulation (POLARIS) methods to obtain the solvation free energies of a series of phenylalanyl peptides with various degrees of methylation on their backbone nitrogens. It was clearly predicted that as a peptide length increased, so solvation free energy decreased, indicating less favorable permeability through the cell membrane system, in agreement with data in the literature. AMSOL also showed that solvation free energy change upon methylation was variable depending on the position of the substituted backbone nitrogen, which disagrees with the literature. However, non-systematic solvation free energy change of small amines upon methylation was successfully predicted by AMSOL, in good accord with experimental data. / Master of Science

The rapid progression of Klebsiella pneumoniae towards antibiotic resistance is a significant concern, primarily due to its protective extracellular polysaccharide (EPS) capsule that shields the bacteria from host immunity. Our previous research demonstrated that antimicrobial peptides could disrupt the EPS capsule of K. pneumoniae. Further analysis identified Bac7 (1-35), a proline-rich antimicrobial peptide (PrAMP), as having the greatest ability to aggregate with the K. pneumoniae EPS capsule, exhibiting potent antimicrobial activity. However, the relationship between key features facilitating EPS and membrane interactions, as well as antimicrobial efficacy, remains poorly understood. Here, we used natural PrAMPs from diverse organisms to investigate their interactions with the cell envelope of K. pneumoniae. Apidaecin Cd3+, Tur1A, and PR-39 peptides demonstrated activity against all tested strains, with a minimum inhibitory concentration ≤ 1 µg/mL. These peptides shared a proline content exceeding 36% and a charge greater than +5. Active PrAMPs induced membrane depolarization in K. pneumoniae, with the extent of depolarization directly correlating with peptide charge, suggesting membrane depolarization as a potential mechanism for PrAMP entry into the cell. Checkerboard assays of active PrAMPs with PepC, an inactive peptide, suggested the membrane actions of PrAMPs have potential to rescue a therapeutic unable to access the bacterial membrane. Consistent with our findings with bac7(1-35) truncated analogs, both active and inactive PrAMPs aggregated with K. pneumoniae EPS, suggesting that the antimicrobial activities and polysaccharide aggregation potential of this class of peptides can be studied independently. Furthermore, the treatment of biofilms with active peptides revealed unique structure-based biofilm changes, with Tur1A causing more structural collapse than PR-39. Our findings highlight a potential membrane mediated peptide uptake into the cell which is dependent on the charge of the peptide. Differential biofilm interactions between similar peptides and EPS aggregation of inactive peptides warrant these attributes of PrAMPs to be further studied independently.

Les liquides ioniques connaissent depuis quelques décennies un essor particulier en raison de leurs nombreuses propriétés physico-chimiques intéressantes, telles qu’une faible pression de vapeur saturante, une viscosité limitée, une faible miscibilité avec la plupart des solvants communs, ou encore des propriétés d’agencement supramoléculaire, qui en font des outils puissants dans de nombreux domaines de la chimie. Les sels d’imidazolium représentent la plus grande famille de liquides ioniques à ce jour. Leur modulabilité leur permet d’être dérivés pour de nombreuses applications spécifiques, notamment en synthèse organique, où ils sont utilisés majoritairement comme solvants, et plus récemment comme catalyseurs. Les travaux présentés dans cette thèse se concentrent sur leur utilisation en synthèse organique, à la fois comme solvants et principalement comme catalyseurs chiraux, catalyseurs pour lesquels l’anion du sel est l’espèce catalytique, permettant d’ajouter de la flexibilité et de la mobilité au système. En tirant parti de la tolérance des liquides ioniques envers la majorité des macromolécules naturelles, l’objectif principal des travaux présentés dans cette thèse est le développement d’un nouveau type de catalyseur bio-hybride reposant sur l’encapsulation d’un sel d’imidazolium dans une protéine. Par le biais de la technologie biotine-avidine, l’inclusion supramoléculaire de sels d’imidazolium biotinylés portant des contre-anions catalytiques dans l’avidine a été réalisée et exploitée en catalyse. Dans un premier temps, le développement et l’étude de deux sels de 1-butyl-3-méthylimidazolium possédant des anions chiraux dérivés de la trans-4-hydroxy-L-proline sont rapportés, ainsi que leur comportement dans des réactions énantiosélectives d’aldol et d’addition de Michael. Ces types de composés se sont révélés actifs et performants en milieu liquide ionique. Dans un second temps, la préparation de sels d’imidazolium dont le cation est biotinylé et portant un contre-anion achiral, a été réalisée. Le comportement de l’avidine en milieu liquide ionique et son apport en termes de chiralité sur le système bio-hybride ont été étudiés. Les résultats montrent le rôle crucial des liquides ioniques sur la conformation de la protéine et l’efficacité du catalyseur pour des réactions d’aldol. Dans un dernier temps, l’influence de la structure du cation et de l’anion sur le système a été étudiée. Différents espaceurs ont été introduits successivement dans les squelettes cationiques et anioniques des sels d’imidazolium biotinylés. Dans le cas du cation, les résultats ne révèlent aucune influence majeure sur l’efficacité du catalyseur. La structure de l’anion se montre cependant beaucoup plus importante : la préparation de différents catalyseurs bio-hybrides possédant des anions aux propriétés physico-chimiques différentes a permis d’obtenir de plus amples informations sur le mode de fonctionnement du système bio-hybride et de la coopérativité entre l’avidine et l’anion du sel d’imidazolium.La nature ionique de la liaison cation-anion offrant une liberté de mouvement accrue à l’anion dans la protéine, la tolérance à différents substrats a également été abordée après optimisation du système. / Ionic liquids have gained a growing interest due to many interesting properties, such as low vapor pressure, reasonably low viscosity, poor miscibility with common organic solvents, and also exhibit supramolecular organization in solution, which make them interesting tools for several fields of applications in chemistry. As of today, imidazolium salts make up the largest family of ionic liquids. Their modulability allows them to be used for a wide range of applications, notably in organic chemistry, where they are mainly used as solvents, but also more recently as actual catalysts. The work presented in this thesis focuses on their use as solvents and chiral catalysts, in which the catalytic species is the anion of the imidazolium salts, adding more flexibility and mobility to the whole system. Taking advantage from the tolerance of ionic liquids toward biological macromolecules, the main goal of this work is the design and development of a new type of bio-hybrid catalyst based on the encapsulation of an imidazolium salt inside the cavity of a host protein. Based on the biotin-avidin technology, the supramolecular ligation of biotinylated imidazolium salts inside avidin, bearing catalytic counter-anion, is discussed. As a first step, the development and studies of two 1-butyl-3-methylimidazolium-based salts, bearing trans-4-hydroxy-L-proline-derived anions are reported. Their use for asymmetric catalysis in ionic liquids media is disclosed, both for the aldol and Michael additions. Results show that these compounds are viable and efficient organocatalysts in ionic liquids. Subsequently, the preparation of biotinylated imidazolium salts, bearing a racemic pyrrolidine-based counter-anion is reported. Avidin behaviour in ionic liquid media, as well as its contribution for the stereocontrol for the whole bio-hybrid system, is assessed. Results highlight the critical role of the ionic liquid reaction medium on the protein’s conformation, and thus the efficiency of the bio-hybrid catalyst towards aldol reactions. Finally, the influence of the structure of the cation and anion on the catalytic properties of the biohybrid system were investigated. Several spacers were inserted successively both in the cation and anion structures of the biotinylated imidazolium salts. Regarding the cation modifications, results show no major influence on the bio-hybrid catalyst behaviour. However, modifying the anion structure revealed the much more important role of the anion towards catalysis. Preparation of different anions, each bearing a different spacer, granting them different physico-chemical properties, gives rise to further information regarding the behaviour of the bio-hybrid catalyst, and possible cooperativity between avidin and the imidazolium salt. The ionic character of the interaction between the anion and the cation, allowing a greater freedom of movement of the anion inside the avidin’s cavity, and the tolerance of the bio-hybrid system to different substrates were studied.

La liaison peptidique au sein d’un peptide ou d’une protéine est d’ordinaire plane et en conformation trans pour la majorité des acides aminés. La situation est un peu différente en amont d’une proline : la barrière thermodynamique qui s’oppose à la rotation de la liaison amide est plus faible et la tendance de la liaison à rester plane est un peu moins grande. Cette liaison AA-Pro peut donc adopter une conformation cis, entraînant la formation d’un coude prononcé dans une chaîne peptidique à son niveau. Ce travail décrit la synthèse et la réactivité chimique de nouveaux analogues bis-azotés de la proline en solution permettant de favoriser la conformation cis d’une liaison AA-ΨPro. L’impact conformationnel que peut engendrer ces résidus au sein de pseudopeptides est également exposé. Dans un premier temps, une nouvelle voie d’accès à la α-azaproline énantiomériquement pure et orthogonalement protégée a été mise au point en exploitant des travaux antérieurs concernant la synthèse d’α-hydrazinoesters et de N-aminodipeptides. L’étude de la réactivité de cette pseudoproline a permis de définir les meilleures conditions de formations de pseudotripeptides de formule P1-AA1-δ-azaPro-AA3-P3. Elle a également orienté les travaux, dans un second temps, vers la synthèse de pseudopeptides incorporant un motif acide pyrazolique. Enfin, la structure des composés préparés a été analysée par RMN, IR et par modélisation moléculaire. L’examen des P1-AA1-δ-azaPro(Boc)-AA3-P3 a révélé la formation par liaison hydrogène d’un pseudocycle en C7, favorisant la conformation trans de la liaison AA1-δ-azaPro, alors que l’absence de la fonction Boc favorise la conformation cis de cette liaison. / The peptidic bond in a peptide or a protein is usually flat and in trans conformation for the majority of amino acids. The situation is a little bit different upstream the proline: the thermodynamic barrier which opposes the rotation of the amide bond is weaker and the tendency of the bond to remain flat is lesser. So, this AA-Pro bond can adopt a cis conformation, leading to the formation of a turn in the peptidic chain. This work describes the synthesis and the chemical reactivity of new bis-nitrogen analogous of proline in solution to facilitate the cis conformation of a AA-PΨPro bond. The conformational impact that these residues may generate in pseudopeptides is also exposed.Initially, a new access road to the orthogonally protected and enantiomerically pure δ-azaproline has been developed by exploiting previous work on the synthesis of α- hydrazinoesters and N-aminodipeptides. The study of the reactivity of this pseudoproline helped define the best conditions for forming pseudotripeptides of formula P1-AA1--δ-azaPro-AA3-P3. It also guided the work, in a second step, towards the synthesis of pseudopeptide incorporating a pyrazole acid motif. Finally, the structure of the prepared compounds was analyzed by NMR, IR and molecular modeling. Examination of the P1-AA1-δ-azaPro(Boc)-AA3-P3 revealed the formation of a pseudocycle C7 by a Hydrogen bond, favoring the trans conformation of the AA1-δ-azaPro bond, while the absence of Boc function seems to favor the cis conformation of this bond.

Le tripeptide GPE possède une activité neuroprotectrice intéressante in-vitro et in-vivo pour différents types de dommage neuronaux. L'objectif de cette thèse est de mettre au point des méthodes de couplage peptidique pour les acides aminés trifluorométhylés et de réaliser des analogues trifluorométhylés du GPE.Au cours de cette thèse, la synthèse d'acides aminés trifluorométhylés cycliques énantiopurs (proline, pseudoproline) a été réalisée à l'échelle du gramme.Ensuite, une étude méthodologique de l'incorporation de ces acides aminés trifluorométhylés dans des peptides a été effectuée.Des expériences RMN ont été menées pour étudier la conformation cis/trans de la liaison amide de ces peptides trifluorométhylés.Enfin, des analogues du tripeptides GPE ont été synthétisé en remplaçant le motif proline par une proline ou pseudoproline trifluorométhylé. Des tests biologiques sont actuellement en cours pour évaluer leur activité sur divers troubles neuronaux. / GPE is a tripeptide with neuroprotecting activity both in-vitro and in-vivo for various neuronal dammage. The goal of this thesis was to work out peptide coupling methods for trifluoromethylated amino acids and the synthesis of trifluoromethylated analogs of GPE.During this thesis, the synthesis of enantiopure cyclic trifluoromethylated amino acids (proline and pseudoprolines) was performed successfully on grams scale.Then, methodological study of their incorporation in peptide was performed.NMR experiments were made to investigate the cis/trans conformation of the amide bound of trifluoromethylated peptide. At last, analogs of the tripeptide GPE were made with trifluoromethylated proline and pseudoprolines instead of the proline. Biological tests are currently under progress to evaluate their activity on various neuronal disorders.

Made available in DSpace on 2016-08-10T10:38:28Z (GMT). No. of bitstreams: 1 Francisco Pereira Borges Filho.pdf: 377994 bytes, checksum: 8fad9d0fa2eb6d066183d54225e6aeab (MD5) Previous issue date: 2009-05-05 / INTRODUCTION: The polymorphism at codon 72, proline (p53P) or arginine (p53R) is involved in the ability of p53 to interact with cellular proteins. Several authors have shown that the presence of genotype p53RR confers greater risk of developing tumors. OBJETIVE: To assess the allelic frequency of genetic polymorphism in codon 72 in TP53 gene in samples obtained from patients diagnosed with squamous carcinoma of base of the tongue treated at the Hospital Araújo Jorge and Associação de Combate ao Câncer em Goiás (ACCG)between 1990 and 2006. Evaluate the genetic predisposition for Base of Tongue Cancer linked of TP53 polimorphism, by identififyin of the presence or absence of the alleles p53R and/or p53P alleles in patients with this pathology. MATERIALS AND METHODS: This study is a casecontrol retrospective in nature, was conducted at Núcleo de Pesquisas Replicon of the Universidade Católica de Goiás in conjunction with the Hospital Araújo Jorge. We evaluated 54 patients with squamous carcinoma of the base of the tongue and 186 individuals without cancer. These were matched regarding gender, age, and the group of cases was evaluated clinical stage and smoking, alcohol consumption. The genotypes p53R and p53P were determined by PCR, using specific primers. RESULTS: The allele frequencies for p53R in cases and controls were 75.9% and 74.2%, while the p53P were 24.1% and 25.8% respectively. There was no statistically significant difference (p = 0.79) in allele frequencies between the two groups, suggesting that the polymorphism of codon 72 of TP53 is not a risk factor for susceptibility to squamous cell carcinoma of the tongue base. CONCLUSION: The study does not find relationship between p53R and CCO carcinogenesis when compared sex, age and color. / INTRODUÇÃO: O polimorfismo no códon 72, prolina (p53P) ou arginina (p53R) está envolvido na habilidade da p53 em interagir com as proteínas celulares. Vários autores têm demonstrado que a presença do genótipo p53RR confere maior risco de desenvolvimento de tumores. OBJETIVO: Avaliar a freqüência alélica do polimorfismo genético no códon 72 do gene p53 em amostras obtidas de pacientes diagnosticados com Carcinoma Escamoso de Base de Língua atendidos no Hospital Araújo Jorge, da Associação de Combate ao Câncer em Goiás (ACCG), entre os anos de 1999 e 2006. Avaliar a predisposição genética do câncer de base da língua relacionada ao polimorfismo de TP53, através da identificação da presença ou não dos alelos p53R e/ou p53P nos pacientes com esta patologia. MATERIAIS E METODOS: O presente estudo é do tipo caso-controle de caráter retrospectivo, foi realizado no Núcleo de Pesquisas Replicon da Universidade Católica de Goiás em conjunto com o Hospital Araújo Jorge. Foram avaliados 54 pacientes com carcinoma escamoso de base da língua e em 186 indivíduos sem câncer. Estes foram pareados quanto ao sexo, idade e no grupo caso, foram avaliados o estádio clínico e hábitos de etilismo e tabagismo. A genotipagem dos alelos p53R e p53P foi determinada por PCR, utilizando-se primers específicos. RESULTADOS: As freqüências alélicas para p53R nos casos e controles foram de 75,9% e 74,2%, enquanto que de p53P foram de 24,1% e 25,8%, respectivamente. Não houve diferença estatisticamente significativa (p = 0,79) nas freqüências alélicas entre os dois grupos analisados, sugerindo que o polimorfismo do códon 72 de TP53 não seja um fator de risco de susceptibilidade ao carcinoma escamoso de base da língua. CONCLUSÃO: Não foi observada associação entre a variante p53R e o desenvolvimento da carcinogênese de CCO de base da língua, segundo o sexo, idade e etnia.

Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) derivatives possess cancerostatic and anti-infective properties thus making them candidates of possible antibacterial agents. ²-lactam antibiotics are vital weapons for the treatment of bacterial infections, but their existence and effectiveness has been faced with resistance from ²-lactamases. Therefore, the need for new effective antimicrobial drugs is very crucial. In this work, we synthesized in high yields, PBD analogs 1−3, 5 and 7−9 in three to four synthetic steps from commercially available L-proline and isatoic anhydride. MTT Assay was employed to test the in vitro cytotoxicity of PBD analogs 1, 2, 5 and 7 on cancer cell lines including MCF-7, SKBR-3, SKMEL-2, CaCo 2 and Mia Paca. These compounds decreased the cell viability of MCF-7 by roughly 20% however, 1 and 5 had no effect on the SKMEL-2 cell lines. The inhibitory efficacy of these PBDs were also tested against TEM-1 and P99 Serine class A and C ²-lactamases.

<p>This thesis describes the design and synthesis of compounds that are</p><p>intended to inhibit serine and aspartic proteases. The first part of the text deals with preparation of inhibitors of the hepatitis C virus (HCV) NS3 serine protease. Hepatitis C is predominantly a chronic disease that afflicts about 170 million people worldwide. The NS3 protease, encoded by HCV, is essential for replication of the virus and has become one of the main targets when developing drugs to fight HCV. The inhibitors discussed here constitute surrogates for the widely used <em>N</em>-acyl-hydroxyproline isostere designated 4-hydroxy-cyclopentene. The stereochemistry of the 4-hydroxy-cyclopentene scaffold was determined by nuclear overhauser effect spectroscopy (NOESY) and the regiochemistry by heteronuclear multiple bond correlation (HMBC). The scaffold was decorated with different substituents to obtain both linear and macrocyclic HCV NS3 protease inhibitors that display low nanomolar activity. The second part of the thesis describes the design and synthesis of potential aspartic protease inhibitors. The hydroxyethylene motif was used as a noncleavable transition state isostere. The synthetic route yielded a pivotal intermediate with excellent stereochemical control, which was corroborated by NOESY experiments. This intermediate can be diversified with different substituents to furnish novel aspartic protease inhibitors.</p> / Report code: LIU-TEK-LIC-2006:45

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.