Thesis (Ph. D.)--Ohio State University, 2008.

Thesis (Ph. D.)--Illinois State University, 1976. / Title from title page screen, viewed Dec. 7, 2004. Dissertation Committee: H.E. Brockman (chair), M. Neville, A. Richardson, F. Schwalm, D. Weber. Includes bibliographical references (leaves 125-138) and abstract. Also available in print.

Thesis (Ph. D.)--Boston University, 1994. / The Escherichia coli mannitol permease (EIIMtl) is a 68 kilodalton (kDa) membrane-bound protein that carries out phosphoenolpyruvate-dependent transport and phosphorylation of D-mannitol. This protein also catalyzes a phosphoexchange reaction between mannitol and mannitol-1-phosphate and acts as a chemotactic receptor for mannitol in this bacterium. The gene that encodes this protein, mtlA, has been cloned and sequenced. A structural model for the EIIMtl has been previously proposed based upon hydropathy analysis of the deduced amino acid sequence of mtlA, proteolysis studies, and 'phoA fusion analysis. According to this model, the N-terminal domain (residues 1-334) is comprised of six or seven membrane-spanning alpha-helices and the C-terminal domain (residues 335-637), which contains two phosphorylation sites, is exposed to the cytoplasm. A series of mtlA deletion mutants was constructed for further analysis of structure/function relationships in the mannitol permease. In this study, several deletion mutants are selected for characterization of their mannitol binding activity, insertion/stability in the membrane, and oligomerization. The results showed that [TRUNCATED]

Indiana University-Purdue University Indianapolis (IUPUI) / Chlamydia trachomatis is the leading cause of bacterial sexually transmitted disease. Chlamydia spp. are all obligate intracellular organisms that undergo a biphasic developmental cycle within a vacuole termed the inclusion. Infectious, non metabolically active elementary bodies (EBs) are endocytosed and differentiate into non infectious, metabolically active reticulate bodies (RBs) before re-differentiating back into EBs. The chlamydial factors that mediate these differentiation events are mostly unknown. Comparative genomics revealed that Chlamydia spp. have small, highly conserved genomes, suggesting that many of their genes may be essential. Genetic manipulation strategies for Chlamydia spp. are in their infancy, and most of these cannot be used to inactivate essential genes. We generated a clonal ethyl methanesulfonate (EMS)-mutagenized C. trachomatis library and screened it for temperature sensitive (TS) mutants that produced fewer inclusions at either 32°C or 40°C compared to 37°C. Because EMS mutagenesis elicited multiple mutations in most of the library isolates, we also developed a novel lateral gene transfer strategy for mapping mutations linked to TS phenotypes. We identified TS alleles of genes that are essential in other bacteria and that are involved in diverse biological processes including DNA replication, protein synthesis, carbohydrate metabolism, fatty acid biosynthesis, and energy generation, as well as in highly conserved chlamydial hypothetical genes. TS DNA polymerase (dnaEts) and glutamyl-tRNA synthestase (gltXts) mutants were characterized further. Both the dnaEts and gltXts mutants failed to replicate their genomes at 40°C but exhibited unique signs of stress. Chlamydial DNA replication begins by 12 hpi and protein synthesis begins by 2 hpi. However, inclusion expansion and replication of both of the mutants could be rescued by shifting to them to 37°C prior to mid-late development. Since gltXts is likely unable to produce aminoacyl-tRNAs at 40°C, our observation suggests that de novo chlamydial translation uses a pre-existing pool of aminoacyl-tRNA in EBs. Genetic suppressor analysis indicated that the inability of the dnaEts mutant to replicate its genome at 40°C might be linked to an inability of mutant DnaE to bind the DNA template. The tools and mutants we have identified will be invaluable assets for investigating many essential aspects of chlamydial biology.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

A high degreee of structural and sequence homology exists between the EGF -1 domains of the vitamin K-dependent coagulation factors, as well as between the EGF-1 domains of individual vitamin K-dependent coagulation factors from various species. Through studies of protein evolution it has been determined the conserved amino acid residues observed are essential for protein structure while the variable residues have been implicated in specific protein-protein interactions. In the case of FVII, this interaction is the high affinity binding of its cofactor, TF, which initiates the extrinsic pathway of coagulation. 43% of the contact area of the FVII molecule in the FVIIa•TF complex is located within the FVII EGF-1 domain. A series of human FVII variants have been constructed in which the EGF-1 domain has been exchanged, either in its entirety or as single amino acid substitutions, with that of the mouse or rabbit. These species were chosen as it had previously been shown that plasma from mouse or rabbit, when combined with human TF, was able to clot at a significantly greater rate than homologous human plasma. We hypothesized that through these exchanges it might be possible to generate a human FVII variant with increased TF binding. Of the FVII variants generated, 2 human FVII point mutants have shown an increased affinity for human TF after transient expression. A75D and T83K exhibited TF binding at 200% and 150%, respectively, of that seen with wild-type human FVII. Both A75D and T83K exhibited clotting and amidolytic activity that was proportionally increased, with respect to TF binding. Computer-generated structures of these variants predict an additional hydrogen bond between the FVII and TF molecules likely to be responsible for the increased TF affinity in the T83K mutant. Intramolecular forces within the FVII molecule are predicted to have caused a conformational change with the A75D mutation which has lead to its increased TF binding. The greater affinity for TF exhibited by the human FVII point mutants A75D and T83K, as compared to wild-type human FVII, is an important step toward the creation of new class of competitive inhibitors of coagulation which are specifically directed towards its initial stage, rather than later in the cascade. However, more study is needed to determine the ability of either of these mutants to compete against each other for TF both 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 and 𝘪𝘯 𝘷𝘪𝘷𝘰. If either mutant is successful, it can then be active site inhibited by site-directed mutagenesis or through the use of chloromethyl ketones, in order to decrease its clotting and amidolytic activity. / Thesis / Master of Science (MSc)

The aim of this study was to investigate the mechanisms of action of and development of cellular resistance to various anticancer agents in human (HeLa) cells using a combined genetic and biochemical approach. The agents employed for this purpose included: the purine nucleoside analogues, toyocamycin, tubercidin, and 6-methyl-mercaptopurine riboside (6-MeMPR); the protein synthesis inhibitor, puromycin; and the microtubule stabilizer, taxol. To investigate the mechanisms of action and cellular resistance to the purine nucleoside analogues, stable first-step toyocamycin, tubercidin and 6-MeMPR resistant HeLa mutants were isolated. These mutants exhibited high degrees of resistance and cross-resistance to various adenosine kinase-activated nucleoside analogues, possessed <2% of the adenosine kinase activity of parental HeLa cell extracts and exhibited severely reduced cellular uptake and macromolecular incorporation of adenosine in vivo. These results indicate that in human cells the cytotoxic effects of toyocamycin, tubercidin and 6-MeMPR are dependent upon adenosine kinase-catalyzed phosphorylation of these drugs to their respective monophosphates and that resistance to these agents results from a deficiency in adenosine kinase activity in vivo. Further insight into the nature of the genetic and biochemical alteration(s) affecting adenosine kinase in these mutants was achieved using SDS-polyacryamide gel electrophoretic and immunoblot analysis. Immunoblots -revealed that each toyocamycin, tubercidin and 6-MeMPR resistant mutant contained similar amounts of cross-reacting material that had the same electrophoretic mobility as adenosine kinase in parental HeLa cells. Therefore, the lesion in these mutants must be a missense type of alteration in the structural gene for adenosine kinase. The utility of the 6-MeMPR resistant mutant selection system for quantitative mutagenesis studies was also investigated. Numerous favourable attributes appropriate to mutagenesis studies were found using this selection system. These included: the obtainment of highly resistant mutants which were stable in the absence of drug, the absence of cell density or cross-feeding effects in the selection system, maximum phenotypic expression required a relatively short time period and mutagen treatment increased the mutant frequency in a linear dose-dependent manner. Thus, selection for genetic alterations at the adenosine kinase locus appears to provide a valuable system for quantitative mutagenesis studies in human cells. The combined genetic and biochemical approach was also used to investigate the development of resistance to puromycin and taxol. Therefore, first-and second-step mutants resistant to each of these drugs were selected and characterized. Cross-resistance and uptake studies with the puromycin resistant mutants suggest that the most common -mechanism for the development of cellular resistance to puromycin in human (HeLa) cells involves an alteration in membrane permeability that reduces drug uptake/transport. Similar studies with the taxol resistant mutants suggest the existence of two possible mechanisms for the development of resistant to this agent in human (HeLa) cells. One mechanism involves a biochemical lesion that specifically affects a microtubule-related cellular component. The second mechanism, however, nonspecifically affects cellular membrane permeability and results in reduced drug uptake/transport. / Thesis / Master of Science (MS)

This thesis is missing a page between pages 60 and 70, and three pages between pages 70 and 81. Since the pages are not all numbered, the specific page numbers cannot be determined. Theses missing pages are not in the other copies of the thesis. -Digitization Centre / Replicate isogenic populations of E. coli were propagated and maintained for over 4000 generations in order to investigate the adaptation of E. coli to increased levels of the mutagen methanesulfonic acid ethyl ester (EMS). Control "C" cell lines were propagated through daily serial culture in the absense of any mutagenic treatment. EMS adapted cell lines "E" / "e" were propagated through daily serial culturing and treated daily with 25Jul of EMS following serial dilution. Mutation frequency and survival assays conducted in this investigation strongly suggest that prior long-term low dose exposure to EMS results in significantly higher levels of resistance to the lethal and mutagenic effects of larger challenge doses of EMS relative to long-term evolved control cell lines "C". In addition, both survival and inhibition disk assays suggest a cross adaptive response between EMS and MNNG, showing enhanced survival and reduced growth inhibition zones in cells adapted to EMS and challenged with MNNG. Preliminary competition experiments suggest relative fitness for the EMS adapted cell lines ( "E" / "e") compared to the "C" control cell lines in both the presence of EMS. Unexpectedly the fitness estimates also suggest a higher relative fitness for the "E" / "e" EMS adapted cell lines in the absence of EMS treatment, suggesting that the EMS specific adaptation may also result in improved fitness in novel environments. Despite the adaptive advantage for the "E" / "e" cell lines suggested by the fitness estimates, the results from the competition experiments are insignificant due to the high degree of variability among replicate fitness estimates. Attempts to induce the adaptive response repair pathway were not successful in either the control "C" or the EMS adapted "E" / "e" cell lines suggesting that enhanced resistance seen in the adapted "E" / "e" cell lines could likely be a result of enhanced activity of the constitutive transferase Ogt and the constitutive glycosylase Tag. The ada and the ogt genes encode the induced and the constitutively-active DNA methyl transeferases in E. coli. As such they appeared to be the most likely candidates for genetic changes responsible for the enhanced resistance to the lethal and mutagenic effects of large doses of alkylating agents in the long-term EMS adapted "E" / "e" cell line. However, the DNA sequences analyzed for the ogt and the ada genes for both the long-term evolved control E. coli cell line "C" and the long-term-evolved EMS adapted "E" / "e" cell line indicate no sequences differences between these two cell lines. Previous studies have primarily observed E. coli's ability to phenotypically acclimate over very short time intervals to EMS. This analysis has shown that long-term genetic adaptation to low doses of EMS results in enhanced resistance to both the lethal and mutagenic effects of larger challenge doses of EMS. / Thesis / Master of Science (MS)

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.