Protein synthesis (mRNA) is tightly regulated under numerous conditions in cardiomyocytes. It can be activated by hormones such as insulin and also by other agents such as phenylephrine (PE) that activates hypertrophy in the heart. Cardiac hypertrophy involves an increase in the muscle mass of the heart, principally in the left ventricular muscle, and the increase is due to enlarged cell size, not increased cell number. A pivotal element of cardiac hypertrophy is an elevation in the rates of protein synthesis, which drives the increase in cell size causing hypertrophy. Unfortunately, we currently lack the understanding of the basic mechanisms that drives hyperactivated protein synthesis. Cardiac hypertrophy is clinically important because it is a major risk factor for heart failure. It initially serves as an adaptive response to increase cardiac output in response to higher demand, but ultimately leads to deterioration of contractility of the heart if hypertrophy is sustained. The main goal of this research project is to understand how hypertrophic agents, such as phenylephrine (PE), activate protein synthesis using adult rat ventricular cardiomyocytes as a model. Specifically, this study focuses on how the translational initiation is controlled by upstream signalling pathways. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

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Rotavirus is the major cause of infantile gastroenteritis. Each year more than 600,000 young children are estimated to die in developing countries throughout the world. Rotavirus infection can be either symptomatic or asymptomatic. But the genetic or molecular basis for rotavirus virulence is not yet clearly understood. NSP4, encoded by genome segment 10, is a multifunctional protein. It is identified as the first viral enterotoxin and is essential for virus morphogenesis and pathogenesis. Analysis of NSP4 from more than 175 strains failed to reveal any sequence motif or amino acid that segregated with the virulence phenotype of the virus. Further, a few studies indicated a lack of consistent correlation between virus virulence and diarrhea inducing ability of the cognate NSP4. To understand the basis for the inconsistency in the enterotoxigenic activity of a few NSP4s reported in a limited number of studies, comparative analysis of the biophysical, biochemical, and biological properties of NSP4ΔN72, which from SA11 and Hg18 was earlier shown to be highly diarrheagenic, from 17 different symptomatic and asymptomatic strains was carried out. To study structure-function relationship we used Thioflavin T fluorescence assay, gel filtration, CD spectroscopy, trypsin susceptibility and enterotoxin assay in newborn mice for all the proteins. Detailed comparative analysis of biochemical and biophysical properties and diarrheagenic activity of the recombinant ΔN72 peptides under identical conditions revealed wide differences among themselves in their resistance to trypsin cleavage, thoflavin T binding, multimerization and conformation without any correlation with their diarrhea inducing abilities. Since earlier studies showed that a secreted peptide (ΔN112) of SA11-NSP4 also induced diarrhea in newborn mice pups, we have generated NSP4ΔN112 deletions from six different strains and tested for their diarrhea inducing ability. The patterns of DD50 values of the ΔN112 peptides was similar to that for ΔN72 peptides, but were 1000-1200-fold less efficient than that of SA11ΔN72. NSP4 exists in multiple forms in the infected cells- as oligomers, higher molecular weight complexes and ER- and cytoplasmic membrane anchored forms. Previous studies suggest that the N-terminal boundary of the oligomerization domain could lie downstream to residue 94 from the N-terminus. A peptide from residue 112-175, secreted from rotavirus infected cells, was reported to induce dose-dependent diarrhea in suckling mice, suggesting that the N-terminal boundary of the enterotoxin activity could lie around residue 112. However, the precise N-terminal boundaries in NSP4 for oligomerization and diarrhea induction have not been identified. To address this question, a large number of deletion mutants C-terminal to residue 94 were generated and tested for their ability to induce diarrhea in newborn mouse pups. Our data suggest that while the deletions ∆N121 to ∆N131 failed to induce diarrhea, ΔN118 was diarrheagenic suggesting that the N-terminal boundary of the minimal diarrhea inducing domain lies between aa 118 and 121. Size exclusion chromatography revealed that residues 95 to 98 are critical and sufficient for oligomerization. Studies on oligomerization further revealed that NSP4ΔN94 exists in pentamers, tetramers and dimers, while deletion mutants C-terminal to aa 94 exist only as dimers. Our studies demonstrate for the first time that not only tetramers but pentamers as well as dimers possess enterotoxigenic properties. Most human rotavirus infections are caused by group A rotaviruses. Within this group, rotaviruses are further classified into subgroups based on the antigenic specificity associated with the protein product of the sixth gene, VP6. Previous studies have mapped SG I specificity to aa position 305 and the region between 296 and 299, and SG II specificity to residue 315 on VP6. However, the subgroup specific determinants on NSP4 have not been identified till date. In this study, we generated several amino acid substitution mutants in the SG I-specific SA11 NSP4∆N72 protein as in previous studies ∆N72 was found to efficiently bind DLPs. Using an enzyme linked immunosorbent assay method, the effect of the mutations in the C-terminal and N-terminal regions in ∆N72 on their binding ability to SG I and SG II DLPs was assayed. Residues at positions 85, 169, 174 and 175 and in the ISVD appear to collectively determine the specificity of binding to DLPs. While the conserved proline and glycines at positions 165, 168 and 162, respectively, are important for maintaining the required conformation for general recognition of DLP. The present study provides important insights towards understanding the determinants in NSP4 for SG-specific DLP interaction.

The work in this thesis focuses on the study of three main aspects of proteins, viz, Protein structure, stability, and dynamics. Chapter 1 is a general introduction to the topics studied in this thesis. Chapter 2 deals with the first aspect, i.e., protein structure in which we describe an approach to use saturation mutagenesis phenotypes to guide protein structure prediction. Chapters 3 and 4 discuss how to increase protein stability using surface electrostatics, and Chapter 5 details a method to predict whether a proline substitution in a given protein would be stabilizing or destabilizing. Hence, Chapters 3-5 can be associated with the second aspect, i.e., protein stability. The third aspect, namely protein dynamics, is dealt with in Chapters 6 and 7 which study conformational dynamics of adenylate kinase. Protein structure prediction is a difficult problem with two major bottlenecks, namely, generation of accurate models and the selection of the most appropriate models from a large pool of decoys. In Chapter 2, the problem of model discrimination is addressed using mutant phenotype information derived from saturation mutagenesis library. A library of ~1500 single-site mutants of the E. coli toxin CcdB (Controller of Cell Division or Death B) has been previously constructed in our lab. The pooled library was characterized in terms of individual mutant phenotypes at various expression levels which were derived from the relative populations of mutants at each expression level. The relative populations of mutants were estimated using deep sequencing. Mutational tolerances were derived from the phenotypic data and were used to define an empirical parameter which correlated with a structural parameter, residue depth. We further studied how this new parameter can be used for model discrimination. Increasing protein stability in a rational way is a challenging problem and has been addressed by various approaches. One of the most commonly used approaches is optimization of protein core residues. Recently, optimization of protein surface electrostatics has been shown to be a useful approach for increasing stability of proteins. In Chapter 3, from analyses of a dataset of ~1750 non-homologues proteins, we show that proteins having a pI away from physiological pH, possess a significant fraction of unfavorably placed charged amino acids on their surface. One way to increase protein stability in such cases might be to alter these surface charges. This hypothesis was validated experimentally by making charge reversal mutations at putative unfavorable positions on the surface of maltose binding protein, MBP. The observed stabilization can potentially be increased by combining multiple individually stabilizing mutations. Different combinations of such mutations were made and tested in Chapter 4 to decide which mutants can be combined to achieve net stabilization. Ideas were tested through systematic experimentation which involved generation of two-site, three-site, and four-site mutations. A maximum increase in melting temperature (Tm) of 3-4 °C over wild-type protein was achieved upon combination of individually stabilizing mutants. Proline (Pro) has two special stereo-chemical properties when it is a part of a polypeptide chain. First the φ value of Pro has a very constrained distribution and second, Pro lacks an amide hydrogen. Due to these properties, introduction of Pro might perturb stability/activity of the protein. In Chapter 5 we describe a procedure to accurately predict the effects of Pro introduction on protein stability. Pro scanning mutagenesis was carried out on the model protein CcdB and the in vivo activity of the individual mutants was also examined. A decision tree was constructed, using the special stereo-chemical properties of Pro to maximize correlation of predicted phenotype with the in vivo activity. Binary classification as perturbing or non-perturbing of every Pro substitution was possible using the decision tree. The performance of the decision tree was assessed on various test systems, and the average accuracy was found to be ~75%. The role of conformational dynamics in enzyme catalysis has been explored in great detail in the literature. In Chapter 6, with the help of very long (350 ns), fully atomistic, explicit solvent molecular dynamics simulations, we studied conformational dynamics of adenylate kinase. We found the existence of a relatively stable state which lies intermediate between the open and closed conformations of the enzyme. The finding was further confirmed by computing a two dimensional configurational free energy surface when motions along each of the two movable domains (LID and NMP) are considered as reaction coordinates. We also discussed possible roles of the intermediate state during enzyme catalysis. The role of water in stabilization of intermediate states was also discussed. In Chapter 7, we studied dynamical coupling between LID and NMP domains of adenylate kinase during domain opening. Our observation suggests that the LID domain should start opening prior to the NMP domain. On the domain opening trajectory, the free energy surface of LID domain was found to be very rugged. We discuss a possible role of water in the ruggedness of the domain motions. The Appendix contains 3 supplementary parts of the thesis. Appendix I is a mutant dataset obtained from 454 sequencing analysis. It includes the normalized number of reads per mutation at each expression level along with mutational sensitivity score. Appendix II is parameters used for one of the electrostatic calculations. Appendix III contains a list of PDB ids used for database analysis in surface electrostatics work discussed in Chapter 3.

Traditional computational protein design (CPD) calculations model sequence perturbations and evaluate their stabilities using a single fixed protein backbone template in an approach referred to as single‐state design (SSD). However, certain design objectives require the explicit consideration of multiple conformational states. Cases where a multistate framework may be advantageous over the single‐state approach include the computer aided discovery of new enzyme substrates, the prediction of protein stabilities, and the design of protein dynamics. These design objectives can be tackled using multistate design (MSD). However, it is often the case that a design objective requires the consideration of a protein state having no available structure information. For such circumstances the multistate framework cannot be applied. In this thesis I present the development of two template and ensemble preparation methodologies and their application to three projects. The purpose of which is to demonstrate the necessary ensemble modeling strategies to overcome limitations in available structure information. Particular emphasis is placed on the ability to recapitulate experimental data to guide modelling of the design space. Specifically, the use of MSD allowed for the accurate prediction of a methyltransferase recognition motif and new substrates, the prediction of mutant sequence stabilities with quantitative accuracy, and the design of dynamics into the rigid Gβ1 scaffold producing a set of dynamic variants whose tryptophan residue exchanges between two conformations on the millisecond timescale. Implementation of both the ensemble, coordinate perturbation followed by energy minimization (PertMin), and template, rotamer optimization followed by energy minimization (ROM), generation protocols developed here allow for exploration and manipulation of the structure space enabling the success of these applications.

A diafiltration technique was used to study drug-protein interactions. Fraction V human serum albumin and plasma and two drugs (phenylbutazone and bishydroxycoumarin) with a high affinity for these substances were used in this investigation. Preliminary experiments were carried out to check for release of foreign substances and for binding of drug to the Amicon diafiltration apparatus. A binding experiment, in the absence of drug, revealed release of a protein-like, ultraviolet absorbing substance from Fraction V human serum albumin. The most suitable method of purification for albumin was by diafiltration with Tris buffer. Binding curves for bishydroxycoumarin - human serum albumin, phenylbutazone - human serum albumin, and bishydroxycoumarin - plasma interactions were obtained. The r and r/Df [subscript omitted] values were calculated and binding parameters estimated by both graphical extrapolation and by a computer non-linear least squares fit analysis. Binding curves were not independent of human serum albumin concentration, but the cause of this effect was not fully resolved. Results showed the diafiltration technique can yield precise data, can be used over a wide macromolecule concentration range and produces a binding curve, from one experiment, over a wide range of molar binding ratios. Use of the Amicon diafiltration apparatus in desorption (washout) experiments and equilibrium or direct experiments was also investigated. Attempts were made to obtain binding data by centrifugation (ultrafiltration) and by a gel filtration technique (Sephadex G-25 batch method). These methods yielded unsatisfactory results which could not be compared with those obtained by diafiltration. This abstract represents the true contents of the thesis submitted. / Pharmaceutical Sciences, Faculty of / Graduate

An accurate plasma concentration-response relationship for propafenone (PF), a potent class 1 antiarrhythmic agent, has not yet been defined. A general pharmacological premise suggests that only the free drug is available to contribute to the observed pharmacological response. It has previously been shown that PF is highly bound to α-l-acid glycoprotein (AAG) which results in a low free PF concentration. The correlation of free PF concentration and response failed to adequately describe the dose response relationship. It has subsequently been shown that upon chronic dosing, two active metabolites, namely 5-hydroxypropafenone (5-OH-PF), and n-depropylpropafenone (n-depropyl-PF) accumulate in humans treated with PF. It is highly likely that the free concentration of PF, in addition to those of 5-OH-PF and n-depropyl-PF, contributes to the observed pharmacological effect following administration of PF at steady-state. To date, no accurate estimation of 5-OH-PF binding in serum has been established. This thesis examines the binding characteristics of PF and 5-OH-PF and their interaction in human serum, and in solutions of AAG, human serum albumin (HSA), high density lipoproteins (HDL), low density lipoproteins (LDL), and very low density lipoproteins (VLDL) using equilibrium dialysis. The binding of PF (2.0 μg/mL) and 5-OH-PF (0.5 μg/mL) was examined in serum when both drug and metabolite were present. The free fraction (FF) of PF and 5-OH-PF in serum was 0.063 ± 0.004 and 0.232 ± 0.020, respectively. Both PF and 5-0H-PF were found to bind to a high affinity, low capacity binding site on AAG, in addition PF showed a second low affinity, high capacity binding site. PF displayed a 10 fold greater affinity for the high affinity binding site on AAG when compared to 5-OH-PF. Both PF and 5-OH-PF showed only one low affinity, high capacity site on HSA of similar affinity. The interaction of PF and 5-OH-PF with HDL, LDL, and VLDL appeared to be due to solubilization, rather than a "true" drug-protein binding interaction, since it correlated well with the concentration of cholesterol within the lipoprotein complex (PF, r²=0.85; 5-OH-PF, r²=0.96). However, PF appeared to show saturable binding to the HDL complex. The uptake of PF and 5-OH-PF was greatest in LDL followed by HDL, and finally VLDL. In serum PF displayed both a high affinity, low capacity, and a low affinity, high capacity binding sites, although a similar observation was expected for 5-OH-PF, only one binding site could be experimentally identified. The uptake of 5-OH-PF by red blood cells (RBC) appeared to be approximately 5 fold greater than that of PF (i.e. The ratio of PF and 5-OH-PF concentration in the red blood cell/plasma was 0.7 ± 0.1 and 3.2 ± 0.5, respectively). When the binding of PF and 5-OH-PF was considered separately, the binding profiles were similar, that is, both drugs showed high affinity binding to AAG, and low affinity binding and/or non-specific binding to other serum proteins such as HSA, HDL, LDL, and VLDL. However, when both drug and metabolite were present, the binding of 5-OH-PF to AAG was found to be reduced. This is thought to occur as a result of the displacement of 5-OH-PF by PF from AAG. Thus, the binding of 5-OH-PF was noted to be more dependent on HSA, and lipoproteins when compared to PF. On the other hand, the binding of PF (2.0 μg/mL), even with the addition of 5-OH-PF, was dependent largely on the concentration of AAG. Although the binding of 5-OH-PF was apparently not altered by the addition of PF in serum, a decrease in the binding of 5-OH-PF by the addition of PF was observed. It is hoped that the understanding gained from this thesis will provide information regarding the relative importance of free PF and 5-OH-PF plasma concentration in future pharmacodynamic studies of PF. / Pharmaceutical Sciences, Faculty of / Graduate

The research carried out within this thesis was aimed at the development and implementation of a biocatalytic route towards Ticagrelor, a platelet-aggregation inhibitor. A bio-retrosynthetic consideration of the target compound yielded different possible strategies, which were analysed in terms of enantioselectivity and efficiency. The ultimate goal was to generate a biocatalyst specifically tailored to the starting material to yield the target compound in high optical purity and conversion. Different approaches to the chemoenzymatic generation of the cyclopropyl subunit (cf figure) in enantiomerically pure form were proposed and tested. The lipase from Thermomyces lanuginosus proved to be the most selective and active enzyme tested and was used as a model enzyme, initially yielding an E of 76 at a conversion of 50% after 48h. Through both reaction engineering and rational protein design approaches the time to attain 50% conversion could be reduced to 24 h while the enantioselectivity of the process increased to 100. Moreover, in a rational protein design approach different residues in the lid of the lipase were identified through analysis of the resolved crystal structures and subsequently mutated in order to investigate the influence of these residues on the overall performance of the lipase towards the target biotransformation. Mutations on Asn88 resulted in the inactivation of the enzyme while an Asp57Asn mutation resulted in a more active enzyme. Ultimately, this research has contributed to making the synthetic route towards Ticagrelor more environmentally sustainable, diminishing the need for the use of toxic, unsustainable and sterically demanding auxiliaries, as well as the amount of waste produced. The principles of green chemistry have been applied to the case studied. The synthetic route towards a key fragment of Ticagrelor has been significantly shortened using a biotransformation with an enzyme that can be recycled and employed in catalytic quantities.

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