abstract: Proteins are essential for most biological processes that constitute life. The function of a protein is encoded within its 3D folded structure, which is determined by its sequence of amino acids. A variation of a single nucleotide in the DNA during transcription (nSNV) can alter the amino acid sequence (i.e., a mutation in the protein sequence), which can adversely impact protein function and sometimes cause disease. These mutations are the most prevalent form of variations in humans, and each individual genome harbors tens of thousands of nSNVs that can be benign (neutral) or lead to disease. The primary way to assess the impact of nSNVs on function is through evolutionary approaches based on positional amino acid conservation. These approaches are largely inadequate in the regime where positions evolve at a fast rate. We developed a method called dynamic flexibility index (DFI) that measures site-specific conformational dynamics of a protein, which is paramount in exploring mechanisms of the impact of nSNVs on function. In this thesis, we demonstrate that DFI can distinguish the disease-associated and neutral nSNVs, particularly for fast evolving positions where evolutionary approaches lack predictive power. We also describe an additional dynamics-based metric, dynamic coupling index (DCI), which measures the dynamic allosteric residue coupling of distal sites on the protein with the functionally critical (i.e., active) sites. Through DCI, we analyzed 200 disease mutations of a specific enzyme called GCase, and a proteome-wide analysis of 75 human enzymes containing 323 neutral and 362 disease mutations. In both cases we observed that sites with high dynamic allosteric residue coupling with the functional sites (i.e., DARC spots) have an increased susceptibility to harboring disease nSNVs. Overall, our comprehensive proteome-wide analysis suggests that incorporating these novel position-specific conformational dynamics based metrics into genomics can complement current approaches to increase the accuracy of diagnosing disease nSNVs. Furthermore, they provide mechanistic insights about disease development. Lastly, we introduce a new, purely sequence-based model that can estimate the dynamics profile of a protein by only utilizing coevolution information, eliminating the requirement of the 3D structure for determining dynamics. / Dissertation/Thesis / Doctoral Dissertation Physics 2016

Les cyanobactéries sont des organismes photosynthétiques capables de convertir le CO₂ en composés organiques et de produire de l’oxygène en utilisant l’énergie lumineuse. Néanmoins, de fortes intensités lumineuses saturent l'appareil photosynthétique, ce qui conduit à la production d'espèces réactives de l'oxygène, dangereuses pour la cellule. Pour y faire face, la photoactive orange carotenoid protein (OCP) induit une dissipation thermique de l’énergie excédentaire récoltée par le complexe d’antennes, le phycobilisome (PBS), afin de diminuer l’énergie arrivant aux centres photochimiques. L'OCP est composé de deux domaines), le domaine C-terminal (CTD) et le domaine N-terminal (NTD), reliés par un domaine de liason flexible (linker). Pendant la photoactivation, le caroténoïde est transféré vers le NTD, les domaines se séparent et le NTD peut interagir avec le PBS. Trois familles d'OCP coexistent (OCPX, OCP1 et OCP2) dans les cyanobactéries modernes. Outre l'OCP, de nombreuses cyanobactéries contiennent également des homologues des domaines OCP, le CTDH et HCP. Les HCP sont une famille de protéines caroténoïdes présentant différents traits photoprotecteurs. La plupart d'entre eux sont de très bons quenchers d'oxygène singulet, et un subclade est capable d'interagir avec le PBS et d'induire une dissipation de l'énergie thermique comme l'OCP. Le rôle de CTDH était inconnu. La présence de ces homologues parallèlement à l'OCP a conforté l'idée générale que l'OCP a une origine évolutive modulaire et que la CTDH et HCP pourraient interagir pour former un complexe OCP-like ayant des caractéristiques et une fonction similaires à celles de l'OCP. Dans cette thèse, je présente la première caractérisation des protéines CTDH. Les CTDH sont des dimères se liant à une molécule de caroténoïde. Le rôle principal de la CTDH est de transférer son caroténoïde au HCP. De plus, les CTDH sont capables de récupérer les caroténoïdes des membranes contrairement aux HCP. Ces résultats suggèrent fortement que les CTDH sont des transporteurs de caroténoïde qui assurent le chargement en caroténoïde sur les HCP. Ce nouveau mécanisme de translocation des caroténoïdes pourrait être multidirectionnel. La résolution de deux structures tridimensionnelles de l'ApoCTDH d'Anabaena a montré que la queue C-terminale du CTDH (CTT) peut adopter différentes conformations. De plus, l'analyse de mutation a démontré que le CTT joue un rôle essentiel dans la translocation des caroténoïdes. Enfin, je rapporte une caractérisation moléculaire du linker reliant les domaines de différents OCP modernes et son rôle au cours de l'évolution de l'OCP. Tout d’abord, j’ai caractérisé les OCP des trois subclades, y compris l’OCPX non caractérisé. OCPX et OCP2 présentent une désactivation rapide par rapport à OCP1. Alors que OCP1 et OCPX peuvent dimériser, OCP2 est stable en tant que monomère. Enfin, j'ai constaté que le linker est essentiel pour la désactivation de l'OCP et qu'il régule la photoactivation. Dans OCP1 et OCPX, le linker ralentit la photoactivation, tandis que dans OCP2, il augmente le taux de photoactivation. L'analyse bioinformatique complète cette caractérisation et fournit une image claire de l'évolution de l'OCP pour répondre efficacement aux conditions de stress. / Cyanobacteria are photosynthetic organisms capable of CO₂ conversion into organic compounds and production of O2 by using light energy. Nevertheless, high light intensities saturate the photosynthetic apparatus leading to production of reactive oxygen species, which are dangerous for the cell. To cope with this, the photoactive Orange Carotenoid Protein (OCP) induces thermal dissipation of the excess energy harvested by the antenna complex, the phycobilisome (PBS) to decrease the energy arriving at the photochemical centers. The OCP is composed of two domains connected by a flexible linker, the C-terminal domain (CTD) and the N-terminal domain (NTD). During photoactivation, the carotenoid is translocated to the NTD, the domains separate and the NTD is able to interact with the PBS. Three OCP families co-exist (OCPX, OCP1 and OCP2) in modern cyanobacteria. In addition to the OCP, many cyanobacteria also contain homologs of OCP domains, the CTDH and HCP. The HCPs are a family of carotenoid proteins with different photoprotective traits. Most of them are very good singlet oxygen quenchers, and one sub-clade is able to interact with the PBS and to induce thermal energy dissipation like OCP. The role of CTDH was unknown. The presence of these homologs in parallel to the OCP supported the general idea that the OCP has a modular evolutionary origin and that the CTDH and HCP can interact forming an OCP-like complex with similar characteristics and function than the OCP.In this thesis, I present the first characterization of the CTDH proteins. CTDHs are dimers binding a carotenoid molecule. The main role of the CTDH is to transfer its carotenoid to the HCP. In addition, CTDHs are able to uptake carotenoids from membranes but not HCPs. These results strongly suggested that the CTDHs are carotenoid carriers that ensure the proper carotenoid loading into HCPs. This novel carotenoid translocation mechanism could be multidirectional. The resolution of two tridimensional structures of the ApoCTDH from Anabaena showed that the C-terminal tail of the CTDH (CTT) can populate different conformations. Moreover, mutational analysis demonstrated that the CTT has an essential role in carotenoid translocation. Finally, I report a molecular characterization of the flexible linker connecting the domains of different modern OCPs and its role during the evolution of the OCP. First, I characterized OCPs from the three subclades, including the uncharacterized OCPX. OCPX and OCP2 present a fast deactivation compared with OCP1. While OCP1 and OCPX can dimerize, OCP2 is stable as monomer. Finally, I found that the linker is essential for the OCP deactivation and it regulates the photoactivation. In OCP1 and OCPX the linker slows down the photoactivation, while in OCP2 it increases the photoactivation rate. Bioinformatic analysis complements this characterization and provides a clear picture of the evolution of the OCP to respond efficiently to stress conditions.

Similarity searches are an essential component to most bioinformatic applications. They form the bases of structural motif identification, gene identification, and insights into functional associations. With the rapid increase in the available genetic data through a wide variety of databases, similarity searches are an essential tool for accessing these data in an informative and productive way. In our chapter, we provide an overview of similarity searching approaches, related databases, and parameter options to achieve the best results for a variety of applications. We then provide a worked example and some notes for consideration. Homology detection is one of the most basic and fundamental problems at the heart of bioinformatics. It is central to problems currently under intense investigation in protein structure prediction, phylogenetic analyses, and computational drug development. Currently discriminative methods for homology detection, which are not readily interpretable, are substantially more powerful than their more interpretable counterparts, particularly when sequence identity is very low. Here I present a computational graph-based framework for homology inference using physiochemical amino acid properties which aims to both reduce the gap in accuracy between discriminative and generative methods and provide a framework for easily identifying the physiochemical basis for the structural similarity between proteins. The accuracy of my method slightly improves on the accuracy of PSI-BLAST, the most popular generative approach, and underscores the potential of this methodology given a more robust statistical foundation.

Analysis of genome sequences is enabling identification of numerous novel characteristics that provide valuable means for genetic and biochemical studies. Of these characteristics, Conserved Signature Indels (CSIs) in proteins which are specific for a given group of organisms have proven particularly useful for evolutionary and biochemical studies. My research work focused on using comparative genomics techniques to identify a large number of CSIs which are distinctive characteristics of fungi and other important groups of organisms. These CSIs were utilized to understand the evolutionary relationships among different proteins (species), and also regarding their structural features and functional significance. Based on multiple CSIs that I have identified for the PIP4K/PIP5K family of proteins, different isozymes of these proteins and also their subfamilies can now be reliably distinguished in molecular terms. Further, the species distribution of CSIs in the PIP4K/PIP5K proteins and phylogenetic analyses of these protein sequences, my work provides important insights into the evolutionary history of this protein family. The functional significance of one of the CSI in the PIP5K proteins, specific for the Saccharomycetaceae family of fungi, was also investigated. The results from structural analysis and molecular dynamics (MD) simulation studies show that this 8 aa CSI plays an important role in facilitating the binding of fungal PIP5K protein to the membrane surface. In other work, we identified multiple highly-specific CSIs in the phosphoketolase (PK) proteins, which clearly distinguish the bifunctional form of PK found in bifidobacteria from its homologs (monofunctional) found in other organisms. Structural analyses and docking studies with these proteins indicate that the CSIs in bifidobacterial PK, which are located on the subunit interface, play a role in the formation/stabilization of the protein dimer. We have also identified 2 large CSIs in SecA proteins that are uniquely found in thermophilic species from two different phyla of bacteria. Detailed bioinformatics analyses on one of these CSIs show that a number of residues from this CSI, through their interaction with a conserved network of water molecules, play a role in stabilizing the binding of ADP/ATP to the SecA protein at high temperature. My work also involved developing an integrated software pipeline for homology modeling of proteins and analyzing the location of CSIs in protein structures. Overall, my thesis work establishes the usefulness of CSIs in protein sequences as valuable means for genetic, biochemical, structural and evolutionary studies. / Dissertation / Doctor of Philosophy (PhD)

PsbO (manganese-stabilizing protein) is the largest extrinsic protein of photosystem II, located on the lumen side of photosystem. It is present in all known oxyphototrophic organisms. PsbO facilitates photosynthetic water splitting, which takes place in an oxygen evolving center (Mn4CaO5 cluster) of photosystem II. This work is focused on PsbO of higher plants and its isoforms, particularly their evolution and functions. Bioinformatic analyses revealed that majority of higher plants express exactly two psbO isoforms. A phylogenetic tree of PsbO sequences has an unusual topology. The two paralogous isoforms do not diverge at the base of the phylogenetic tree, as anticipated, but rather at the end of particular branches, at the level of family or lower taxonomic unit. In this work we propose and discuss several hypotheses concerning evolution of PsbO isoforms. The work further includes detailed analysis and identification of protein spots assigned to PsbO on 2D IEF-SDS PAGE gels of potato thylakoid proteins. We identified predominant version of PsbO isoform in most of the spots. We did not succeed to find any posttranslational modification. We optimized a method of psbO expression in E. coli and subsequent purification, which yielded relatively big amount of properly folded recombinant protein. Analysis of...

An infinitesimal fraction of the practically infinite sequence space has achieved enormous functional diversity of proteins during evolution. Intrinsically disordered proteins (IDPs) which lack a fully defined three-dimensional structure are the most likely precursors to today's proteins because of their flexible conformation and functional diversity. But how have these proteins evolved into often rigid and highly specialized protein structures? This evolutionary trajectory has the greatest support in the theory of induced fold whereby the development of the structure was mediated by the interaction and coevolution of primordial unstructured proteins with different cofactors or RNA molecules. Although some random sequences from the sequence space which is not used by nature are also able to form folded proteins the more suitable candidates for evolution of structure and function appear to be random sequences with a high content of disordered which have low aggregation propensity. The selected random protein sequences with high disorder content have been structurally characterized in this work for their further use in evolutionary studies. Three artificial proteins were selected from a random-sequence library based on previous study in our laboratory. In the present work they were purified and...

abstract: Traditionally, allostery is perceived as the response of a catalytic pocket to perturbations induced by binding at another distal site through the interaction network in a protein, usually associated with a conformational change responsible for functional regulation. Here, I utilize dynamics-based metrics, Dynamic Flexibility Index and Dynamic Coupling Index to provide insight into how 3D network of interactions wire communications within a protein and give rise to the long-range dynamic coupling, thus regulating key allosteric interactions. Furthermore, I investigate its role in modulating protein function through mutations in evolution. I use Thioredoxin and β-lactamase enzymes as model systems, and show that nature exploits "hinge-shift'' mechanism, where the loss in rigidity of certain residue positions of a protein is compensated by reduced flexibility of other positions, for functional evolution. I also developed a novel approach based on this principle to computationally engineer new mutants of the promiscuous ancestral β-lactamase (i.e., degrading both penicillin and cephatoxime) to exhibit specificity only towards penicillin with a better catalytic efficiency through population shift in its native ensemble.I investigate how allosteric interactions in a protein can regulate protein interactions in a cell, particularly focusing on E. coli ribosome. I describe how mutations in a ribosome can allosterically change its associating with magnesium ions, which was further shown by my collaborators to distally impact the number of biologically active Adenosine Triphosphate molecules in a cell, thereby, impacting cell growth. This allosteric modulation via magnesium ion concentrations is coined, "ionic allostery''. I also describe, the role played by allosteric interactions to regulate information among proteins using a simplistic toy model of an allosteric enzyme. It shows how allostery can provide a mechanism to efficiently transmit information in a signaling pathway in a cell while up/down regulating an enzyme’s activity. The results discussed here suggest a deeper embedding of the role of allosteric interactions in a protein’s function at cellular level. Therefore, bridging the molecular impact of allosteric regulation with its role in communication in cellular signaling can provide further mechanistic insights of cellular function and disease development, and allow design of novel drugs regulating cellular functions. / Dissertation/Thesis / Doctoral Dissertation Physics 2020

L’évolution des protéines est un domaine important de la recherche en bioinformatique et catalyse l'intérêt de trouver des outils d'alignement qui peuvent être utilisés de manière fiable et modéliser avec précision l'évolution d'une famille de protéines. TM-Align (Zhang and Skolnick, 2005) est considéré comme l'outil idéal pour une telle tâche, en termes de rapidité et de précision. Par conséquent, dans cette étude, TM-Align a été utilisé comme point de référence pour faciliter la détection des autres outils d'alignement qui sont en mesure de préciser l'évolution des protéines. En parallèle, nous avons élargi l'actuel outil d'exploration de structures secondaires de protéines, Helix Explorer (Marrakchi, 2006), afin qu'il puisse également être utilisé comme un outil pour la modélisation de l'évolution des protéines. / Protein evolution is an important field of research in bioinformatics and catalyzes the requirement of finding alignment tools that can be used to reliably and accurately model the evolution of a protein family. TM-Align (Zhang and Skolnick, 2005) is considered to be the ideal tool for such a task, in terms of both speed and accuracy. Therefore in this study, TM-Align has been used as a point of reference to facilitate the detection of other alignment tools that are able to accurately model protein evolution. In parallel, we expand the existing protein secondary structure explorer tool, Helix Explorer (Marrakchi, 2006), so that it can also be used as a tool to model protein evolution.

Protein evolution is a complex and diverse process, yielding an incredible assortment of biological functions and pathways occurring in the cells of living organisms. The way in which a protein's structure is constrained by its functional role and its notable conservation across even distant evolutionary relationships highlight structure as an important unit when considering the evolutionary dynamics of proteins. This thesis attempts to place the structural landscape of the protein universe within an evolutionary framework. We investigate potential evolutionary histories of protein superfamilies by introducing an age, which estimates when the ancestor of that superfamily first evolved. The range of ages of known protein superfamilies goes right back to those which evolved before the diversification of life into three major superkingdoms. The structures of these proteins are varied but those which have evolved more recently tend to be shorter and have a less elaborate globular packing. Protein structures sit within a complex global landscape of three-dimensional folds and we attempt to model the dynamics of this space using networks of folds. These networks consist of a structurally diverse core of folds with older ages, and neighbouring folds tend to be of similar ages. Moreover, there are a few pivotal folds which appear repeatedly as central in the landscapes, connecting together otherwise disparate portions of the space. Sequence profiles which capture patterns of conservation and variation amongst naturally occurring proteins within a superfamily can be compared to identify distant evolutionary relationships. The power of these profiles to detect such relationships is improved by seeding them with structural alignments. A landscape of evolutionary links crossing between different protein folds is presented.

Made available in DSpace on 2014-06-11T19:30:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-09-28Bitstream added on 2014-06-13T19:19:35Z : No. of bitstreams: 1 silva_rht_dr_sjrp.pdf: 929264 bytes, checksum: 5ac0a998512f2430142616c64c6de249 (MD5) / Efeito das mutações sobre a estabilidade das proteínas e uma questão crucial na evolução da proteína. Tais efeitos dependem fortemente do car ater hidrofóbico global da proteína. Em um trabalho recente (J. Chem. Phys.125,084904(2006), n os sugerimos dois cenários de enovelamento com consequências distintas ma evolução da proteína. O limite de baixa hidrofobicidade, corresponde ao regime em que ocorre concomitantemente o colapso e a formação da estrutura nativa. Sob estas condições as proteínas são pouco robustas a mutações, o que implica em uma alta homologia entre proteínas de diferentes espécies. O limite de alta hidrofobicidade, corresponde ao regime em que a proteína sofrem um colapso antes do enovelamento, e neste caso as proteínas são mais robustas a proteínas, sugerindo uma menor homologia entre proteínas de diferentes espécies. Neste trabalho, n os estudamos a homologia de quatro proteínas para 41 espécies diferentes, correlacionando as suas homologias com suas hidrofobicidades médias. As proteínas estudadas foram lisozima, citocromo-c, mioglobina e histona H3, utilizando seis escalas hidrofóbicas diferentes. Junto com o cálculo da homologia, foi realizada uma comparação da similaridade estrutural (rmsd). Os resultados con rmam a hipótese acima, indicando que proteínas, em condições de baixa hidrofobicidade, têm baixa variabilidade de sequências e conformações, para alta hidrofobicidade, as proteínas exibem variabilidade de sequências e conformações / Efect of mutations on stability of proteins is a crucial issue in protein evolution. Such e ects depend strongly on the overall hydrophobic protein character. In a recent work we suggested two scenarios for folding with distinct protein evolution consequences (J. Chem. Phys.125 084904,2006) Under low hydrophobic conditions proteins collapse concomitantly with the formation of their native state, and are less robust to mutations, which implies higher homology among proteins of di erent species. On the other limit, at high hydrophobicity proteins collapse before folding, and in this case they are more susceptible to mutations, suggesting lower homology among proteins of di erent species. In this work we investigate this conjecture studying the homology of four proteins for 41 di erent species, correlating it with their average hydrophobicity. The proteins studied were lysozyme, cytochrome-c, myoglobin and histone H3, using six di erent hydrophobic scales. Along with the homology calculation, a comparison of structural similarity (rmsd) was also carried out. The results con rm the above hypothesis, indicating that proteins at low hydrophobicity display low variations on sequences and conformations. On other hand, at high hydrophobicity, proteins exhibit high variability on sequences and conformations. Keywords: evolution, protein folding, scenarios of folding, projetabilidade, hydrophobicity, homology of proteins, mutations in proteins