Computational methods of analyzing, simulating, and modeling proteins are essential towards understanding protein structure and its interactions. Computational methods are easier as not all protein structures can be determined experimentally due to the inherent difficultly of working with some proteins. In order to predict, design, analyze, simulate or model a protein, data from experimentally determined proteins such as those located in the repository of the Protein Data Bank (PDB) are essential. The assumption here is that we can use pieces of known proteins to piece together a "new" protein hence, de novo protein design. The analysis of the geometric relationships between secondary structure elements in proteins can be extremely useful to protein prediction, analysis, and de novo design. This thesis project involves creating a database of protein secondary structure elements and geometric information for rapid protein assembly, de novo protein design, prediction and analysis.

<p>RNA, or ribonucleic acid, is one of the three biological macromolecule types essential for all known life forms, and is a critical part of a variety of cellular processes. The well known functions of RNA molecules include acting as carriers of genetic information in the form of mRNAs, and then assisting in translation of that information to protein molecules as tRNAs and rRNAs. In recent years, many other kinds of non-coding RNAs have been found, like miRNAs and siRNAs, that are important for gene regulation. Some RNA molecules, called ribozymes, are also known to catalyze biochemical reactions. Functions carried out by these recently discovered RNAs, coupled with the traditionally known functions of tRNAs, mRNAs, and rRNAs make RNA molecules even more crucial and essential components in biology.</p><p>Most of the functions mentioned above are carried out by RNA molecules associ- ating themselves with proteins to form Ribonucleoprotein (RNP) complexes, e.g. the ribosome or the splicesosome. RNA molecules also bind a variety of small molecules, such as metabolites, and their binding can turn on or off gene expression. These RNP complexes and small molecule binding RNAs are increasingly being recognized as potential therapeutic targets for drug design. The technique of computational structure-based rational design has been successfully used for designing drugs and inhibitors for protein function, but its potential has not been tapped for design of RNA or RNP complexes. For the success of computational structure-based design, it is important to both understand the features of RNA three-dimensional structure and develop new and improved algorithms for protein and RNA design.</p><p>This document details my thesis work that covers both the above mentioned areas. The first part of my thesis work characterizes and analyzes RNA three-dimensional structure, in order to develop new methods for RNA validation and refinement, and new tools for correction of modeling errors in already solved RNA structures. I collaborated to assemble non-redundant and quality-conscious datasets of RNA crystal structures (RNA09 and RNA11), and I analyzed the range of values occupied by the RNA backbone and base dihedral angles to improve methods for RNA structure correction, validation, and refinement in MolProbity and PHENIX. I rebuilt and corrected the pre-cleaved structure of the HDV ribozyme and parts of the 50S ribosomal subunit to demonstrate the potential of new tools and techniques to improve RNA structures and help crystallographers to make correct biological interpretations. I also extended the previous work of characterizing RNA backbone conformers by the RNA Ontology Consortium (ROC) to define new conformers using the data from the larger RNA11 dataset, supplemented by ERRASER runs that optimize data points to add new conformers or improve cluster separation.</p><p>The second part of my thesis work develops novel algorithms for structure-based</p><p>protein redesign when interactions between distant residue pairs are neglected and the design problem is represented by a sparse residue interaction graph. I analyzed the sequence and energy differences caused by using sparse residue interaction graphs (using the protein redesign package OSPREY), and proposed a novel use of ensemble-based provable design algorithms to mitigate the effects caused by sparse residue interaction graphs. I collaborated to develop a novel branch-decomposition based dynamic programming algorithm, called BWM*, that returns the Global Minimum Energy Conformation (GMEC) for sparse residue interaction graphs much faster than the traditional A* search algorithm. As the final step, I used the results of my analysis of the RNA base dihedral angle and implemented the capability of RNA design and RNA structural flexibility in osprey. My work enables OSPREY to design not only RNA, but also simultaneously design both the RNA and the protein chains in a RNA-protein interface.</p> / Dissertation

Domain swapping is a wide spread phenomenon which involves the association between two or more protein subunits such that intra-molecular interactions between domains in each subunit are replaced by equivalent inter-molecular interactions between the same domains in different subunits. This thesis is devoted to the analysis of the factors that drive proteins to undergo such association modes. The specific system analyzed is the monomer to swapped dimer formation of the B1 domain of the immunoglobulin G binding protein (GB1). The formation of this dimer was shown to be fostered by 4 amino acid substitutions (L5V, F30V, Y33F, A34F) (Byeon et al., 2003). In this work, computational protein design and molecular dynamics simulations, both with detailed atomic models, were used to gain insight into how these 4 mutations may promote the domain swapping reaction. The stability of the wt and quadruple mutant GB1 monomers was assessed using the software DESIGNER, a fully automatic procedure that selects amino acid sequences likely to stabilize a given backbone structure (Wernisch et al., 2000). Results suggest that 3 of the mutations (L5V, F30V, A34F) have a destabilizing effect. The first mutation (L5V) forms destabilizing interactions with surrounding residues, while the second (F30V) is engaged in unfavorable interactions with the protein backbone, consequently causing local strain. Although the A34F substitution itself is found to contribute favorably to the stability of the monomer, this is achieved only at the expense of forcing the wild type W43 into a highly strained conformation concomitant with the formation of unfavorable interactions with both W43 and V54. Finally, we also provide evidence that A34F mutation stabilizes the swapped dimer structure. Although we were unable to perform detailed protein design calculations on the dimer, due to the lower accuracy of the model, inspection of its 3D structure reveals that the 34F side chains pack against one another in the core of the swapped structure, thereby forming extensive non-native interactions that have no counterparts in the individual monomers. Their replacement by the much smaller Ala residue is suggested to be significantly destabilizing by creating a large internal cavity, a phenomenon, well known to be destabilizing in other proteins. Our analysis hence proposes that the A34F mutation plays a dual role, that of destabilizing the GB1 monomer structure while stabilizing the swapped dimer conformation. In addition to the above study, molecular dynamics simulations of the wild type and modeled quadruple mutant GB1 structures were carried out at room and elevated temperatures (450 K) in order to sample the conformational landscape of the protein near its native monomeric state, and to characterize the deformations that occur during early unfolding. This part of the study was aimed at investigating the influence of the amino acid sequence on the conformational properties of the GB1 monomer and the possible link between these properties and the swapping process. Analysis of the room temperature simulations indicates that the mutant GB1 monomer fluctuates more than its wild type counter part. In addition, we find that the C-terminal beta-hairpin is pushed away from the remainder of the structure, in agreement with the fact that this hairpin is the structural element that is exchanged upon domain swapping. The simulations at 450 K reveal that the mutant protein unfolds more readily than the wt, in agreement with its decreased stability. Also, among the regions that unfold early is the alpha-helix C-terminus, where 2 out of the 4 mutations reside. NMR experiments by our collaborators have shown this region to display increased flexibility in the monomeric state of the quadruple mutant. Our atomic scale investigation has thus provided insights into how sequence modifications can foster domain swapping of GB1. Our findings indicate that the role of the amino acid substitutions is to decrease the stability of individual monomers while at the same time increase the stability of the swapped dimer, through the formation of non-native interactions. Both roles cooperate to foster swapping.

Post-translational modification (PTM) of proteins by enzymes such as methyltransferases, kinases and deacetylases play a crucial role in the regulation of many metabolic pathways. Determining the substrate scope of these enzymes is essential when studying their biological role. However, the combinatorial nature of possible protein substrate sequences makes experimental screening assays intractable. To predict new substrates for proteins, various computational approaches have been developed. Our method relies on crystallographic data and a novel multistate computational protein design algorithm. We previously used our method to successfully predict four new substrates for SMYD2 (Lanouette S & Davey J.A., 2015), doubling the number of known targets for this PTM enzyme that has been difficult to characterize using other methods. This was possible by first extracting a specificity profile of Smyd2 using our algorithm and subsequently screening a peptide library for matching sequences. However, our method did not yield successful results when attempting to reproduce specificity profiles of other proteins (64% accuracy on average). Different protein environments have demonstrated limitations in the methodology and lead us to further develop the algorithm on a more thorough dataset. Using our new optimized method, specificity profile predictions increase by roughly 20% (84% accuracy on average), independent of the structural template used. The algorithm was then used to blindly predict a specificity profile for the methyltransferase Smyd3, an enzyme for which limited data is currently available. A library of 2550 peptides was screened with the predicted profile, yielding 123 matching sequences. We randomly chose 64 for experimental validation (SPOT peptide array) of methylation by Smyd3 and found 45 methylated and 19 non-methylated peptides (70% success rate). Finally, we released to the community a web version of the algorithm, which can be accessed as http://viper.science.uottawa.ca.

The plant homeodomain (PHD) is a protein domain of ~45�100 residues characterised by a Cys4-His-Cys3 zinc-binding motif. When we commenced our study of the PHD in 2000, it was clear that the domain was commonly found in proteins involved in transcription. Sequence alignments indicate that while the cysteines, histidine and a few other key residues are strictly conserved, the rest of the domain varies greatly in terms of both amino acid composition and length. However, no structural information was available on the PHD and little was known about its function. We were therefore interested in determining the structure of a PHD in the hope that this might shed some light on its function and molecular mechanism of action. Our work began with the structure determination of a representative PHD, Mi2b-P2, and this work is presented in Chapter 3. Through comparison of this structure with the two other PHD structures that were determined during the course of our work, it became clear that PHDs adopt a well-defined globular fold with a superimposable core region. In addition, PHDs contain two loop regions (termed L1 and L3) that display increased flexibility and overlay less well between the three PHD structures available. These L1 and L3 regions correspond to variable regions identified earlier in PHD sequence alignments, indicating that L1 and L3 are probably not crucial for the PHD fold, but are instead likely to be responsible for imparting function(s) to the PHD. Indeed, numerous recent functional studies of PHDs from different proteins have since demonstrated their ability in binding a range of other proteins. In order to ascertain whether or not L1 and L3 were in fact dispensable for folding, we made extensive mutations (including both insertions and substitutions) in the loop regions of Mi2b-P2 and showed that the structure was maintained. We then went on to illustrate that a new function could be imparted to Mi2b-P2 by inserting a five-residue CtBP-binding motif into the L1 region and showed this chimera could fold and bind CtBP. Having established that the PHD could adopt a new binding function, we next sought to use combinatorial methods to introduce other novel functions into the PHD scaffold. Phage display was selected for this purpose, because it is a well-established technique and has been used successfully to engineer zinc-binding domains by other researchers. However, in order to establish this technique in our laboratory, we first chose a control system in which two partner proteins were already known to interact in vitro. We chose the protein complex formed between the transcriptional regulators LMO2 and ldb1 as a test case. We have examined this interaction in detail in our laboratory, and determined its three-dimensional structure. Furthermore, inappropriate formation of this complex is implicated in the onset of T-cell acute lymphoblastic leukemia. We therefore sought to use phage display to engineer ldb1 mimics that could potentially compete against wild-type ldb1 for LMO2, and this work is described in Chapter 4. Using a phage library containing ~3 x 10 7 variants of the LMO2-binding region of ldb1, we isolated mutants that were able to interact with LMO2 with higher affinity and specificity than wild-type ldb1. These ldb1 mutants represent a first step towards finding potential therapeutics for treating LMO-associated diseases. Having established phage display in our laboratory, we went on to search for PHD mutants that could bind selected target proteins. This work is described in Chapter 5. We created three PHD libraries with eight randomized residues in each of L1, L3 or in both loops of the PHD. These PHD libraries were then screened against four target proteins. After four rounds of selection, we were able to isolate a PHD mutant (dubbed L13-FH6) that could bind our test protein Fli-ets. This result demonstrates that a novel function can be imparted to the PHD using combinatorial methods and opens the way for further work in applying the PHD scaffold to other protein design work. In summary, the work detailed in Chapters 3 and 5 demonstrates that the PHD possesses many of the properties that are desirable for a protein scaffold for molecular recognition, including small size, stability, and a well-characterised structure. Moreover, the PHD motif possesses two loops (L1 and L3) of substantial size that can be remodeled for target binding. This may lead to an enhancement of binding affinities and specificities over other small scaffolds that have only one variable loop. In light of the fact that PHDs are mainly found in nuclear proteins, it is reasonable to expect that engineered PHDs could be expressed and function in an intracellular environment, unlike many other scaffolds that can only function in an oxidizing environment. Therefore, our results together with other currently available genomic and functional information indicate PHD is an excellent candidate for a scaffold that could be used to modify cellular processes. Appendices 1 and 2 describe completed bodies of work on unrelated projects that I have carried out during the course of my PhD candidature. The first comprises the invention and application of DNA sequences that contain all N-base sequences in the minimum possible length. This work is presented as a reprint of our recently published paper in Nucleic Acids Research. The second Appendix describes our structural analysis of an antifreeze protein from the shorthorn sculpin, a fish that lives in the Arctic and Antarctic oceans. This work is presented as a manuscript that is currently under review at the Journal of the American Chemical Society.

La conception des protéines ou ‘protein design' a pour but de développer des protéines possédant de nouvelles caractéristiques structurales et/ou fonctionnelles. Le principe consiste à identifier parmi toutes les séquences compatibles avec le repliement d'intérêt, celles qui vont conférer à la protéine, la fonction désirée. La procédure générale est réalisée en deux étapes. La première consiste à calculer une matrice d'énergie contenant les énergies d'interactions entre toutes les paires de résidus de la protéine en autorisant successivement tous les types d'acides aminés dans toutes leurs conformations possibles. La seconde étape, ou ‘phase d'optimisation', consiste à explorer simultanément l'espace des séquences et des conformations afin de déterminer la combinaison optimale d'acides aminés étant donné le repliement de départ. Ensuite, différents filtres peuvent être appliqués pour sélectionner les séquences fonctionnelles (étant donné le repliement d'intérêt) des non fonctionnelles. La première étape a consisté au développement de la procédure de ‘protein design', en particulier, à la mise en place et à l'optimisation de la fonction d'énergie ainsi qu'à l'implémentation de l'algorithme d'optimisation. Nous avons montré que notre procédure est robuste puisqu'elle a fait ses preuves dans des applications très diverses telles que la prédiction de l'orientation des chaînes latérales, la prédiction des changements de stabilité ou d'affinité associés à des mutations ponctuelles, ou encore la production de séquences de type natif pour un jeu de protéines globulaires. Pour l'ensemble de ces applications, la qualité des résultats obtenus est comparable à celle observée chez d'autres groupes. Ensuite, nous avons appliqué notre procédure à des systèmes plus complexes tels que les systèmes protéine:ligand. Nous nous sommes intéressés à l'aspartyl-ARNt synthétase (AspRS) et l'asparaginyl-ARNt synthétase (AsnRS). Ces enzymes jouent un rôle crucial dans la traduction du code génétique. Les synthétases fixent leur acide aminé spécifique sur leur ARNt correspondant établissant ainsi l'intégrité du code génétique. Tout d'abord nous avons réalisé le ‘design' des sites actifs d'AspRS et d'AsnRS en présence de leur ligand natif et non natif afin d'évaluer les performances de notre procédure. La qualité des séquences prédites est comparable à celle observée pour les protéines globulaires entières. Par ailleurs, nous avons montré que notre procédure était sensible à la nature du ligand présent dans la poche. Enfin, nous avons réalisé le ‘design' d'un nombre limité de positions dans le site actif de l'AsnRS de façon à ce qu'elle lie préférentiellement l'aspartate au détriment de l'asparagine. Un jeu de mutants prometteurs fut retenu. Leur stabilité et affinité pour les ligands natifs et non natifs est actuellement analysé par des simulations de dynamique moléculaire.

A highly combinatorial structure-based protein engineering method for obtaining enantioselectivity is reported that results in a thorough modification of the substrate binding pocket of Candida antarctica lipase A (CALA). Nine amino acid residues surrounding the entire pocket were simultaneously mutated, contributing to a reshaping of the substrate pocket to give increased enantioselectivity and activity for a sterically demanding substrate. This approach seems to be powerful for developing enantioselectivity when a complete reshaping of the active site is required. Screening toward ibuprofen ester 1, a substrate for which previously used methods had failed, gave variants with a significantly increased enantioselectivity and activity. Wild-type CALA has a moderate activity with an E value of only 3.4 toward this substrate. The best variant had an E value of 100 and it also displayed a high activity. The variation at each mutated position was highly reduced, comprising only the wild type and an alternative residue, preferably a smaller one with similar properties. These minimal binary variations allow for an extremely condensed protein library. With this highly combinatorial method synergistic effects are accounted for and the protein fitness landscape is explored efficiently.

Using protein design, a series of metal binding proteins have been designed, allowing the local factors that contribute to metal affinity and thermostability to be studied. Those proteins with the highest metal binding affinities had the lowest apo-form Tm and the largest ÄTm upon metal binding. In this thesis, major steps have been taken toward applying the engineered protein to MR imaging. The progress of magnetic resonance imaging is hindered by low specificity and rapid elimination of FDA-approved MRI contrast agents. The engineered protein contrast agent has been conjugated to a cancer-specific targeting peptide and encapsulated in albumin microspheres to provide tandem passive and active tumor targeting. Also, a simple, high-yield purification method has been developed.

<p>Biosensors incorporating proteins as molecular recognition elements for analytes are used in clinical diagnostics, as biological research tools, and to detect chemical threats and pollutants. This work describes the application of protein engineering techniques to address three aspects in the design of protein-based biosensors; the transduction of binding into an observable, the manipulation of affinities, and the diversification of specificities. The periplasmic glucose-binding protein from the hyperthermophile Thermotoga maritima (tmGBP) was fused with green fluorescent protein variants to construct a fluorescent ratiometric sensor that is sufficiently robust to detect glucose up to 67°C. Ligand-binding affinities of tmGBP were changed by altering a C-terminal helical domain that tunes ligand binding affinity through conformational coupling effects. This method was extended to the Escherichia coli arabinose-binding protein. Computational design techniques were used to diversify the specificity of the E. coli maltose-binding protein (ecMBP) to bind ibuprofen, a non-steroidal antiinflammatory drug. These designs ranged in affinity from 0.24 to 0.8 mM and function as reagentless fluorescent sensors. The ligand affinities of ecMBP are tuned by complex interactions that control conformational coupling. These experiments demonstrate that long-range conformational effects as well as molecular recognition interactions need to be considered in the design of high-affinity receptors.</p> / Dissertation

Threefoil is a small engineered protein of 141 amino acids which is a member of the beta-trefoil superfamily, with three-fold symmetry and high thermal and kinetic stability. Its primary sequence was designed based on a predicted beta-trefoil glycosidase from the halophilic Archaeon Haloarcula marismortui. Threefoil predominantly forms inclusion bodies when over-expressed in Escherichia coli at 37??C, with little to no protein soluble in the cytoplasm. Nevertheless, Threefoil is capable of adopting a native beta-trefoil structure when refolded from solubilized inclusion bodies. The focus of this thesis is on characterization of the folding and stability of Threefoil through thermodynamic and kinetic experiments for wild-type Threefoil, in addition to sugar- and metal-binding studies and characterization of Threefoil inclusion bodies. Various Threefoil mutants, designed to increase protein stability, are also characterized to probe the origins of, as well as to give insight into, the mechanism of inclusion body formation. The thermodynamic and kinetic stability of wild-type Threefoil was studied using spectral probes, mainly fluorescence, circular dichroism (CD) and dynamic light scattering (DLS). The major observed spectral changes in kinetic and thermodynamic experiments can be fit to a 2-state transition between the folded state and a denatured state containing extensive residual secondary structure. At high protein concentrations, the folding of wild-type Threefoil is complicated by protein misfolding and aggregation. As Threefoil is remarkably resistant to denaturation even at high concentrations of urea and guanidine hydrochloride (GuHCl), studies were also conducted in guanidine isothiocyanate (GuSCN), which is a much stronger denaturant than urea and GuHCl. Remarkably, the time that is required for Threefoil samples to reach equilibrium in renaturation curves is approximately 100 days, while equilibrium by denaturation in the stronger denaturant, GuSCN, requires more than two years. The expression levels of Threefoil mutants A62V, Q78I, D85P and D93P were also studied. None of the four mutants studied exhibited any pronounced increase in solubility compared to wild-type when expressed in E. coli.