The last few decades have witnessed an upsurge in the availability of large-scale data on genomes and genome-scale information. The development of methods to understand the trends and patterns from large scale data promised potentially to unravel the mechanisms responsible for the enormous diversity observed in biological systems. Of the many mechanisms adopted, protein-protein interactions represent one of the commonly adopted mechanisms to achieve functional diversity using a limited genetic repertoire. Protein-proteins interactions bring about several fundamental cellular processes and also modulate regulation at the cellular level. Different types of protein-protein interactions have evolved to carry out myriad functions in a cell. Mainly, interactions can be permanent or transient in nature, depending on the duration of interaction. In terms of affinity ,they are classified as obligate or non-obligate interactions. Structural studies on the various kinds of complexes have enabled the identification of the distinctive features characterizing the different types of complexes. Further, identifying the mechanisms involved in the evolution of protein-protein interactions are important in understanding the forces involved in their maintenance. Such studies also provide clues for the development of methods to predict protein-protein interactions from the large repertoire of sequence and structural data available. In spite of significant understanding of various aspects of protein-protein interactions, several questions still remain unanswered. The work embodied in this thesis studies two main aspects of protein-protein interactions for various types of complexes: structural and evolutionary features. The first part of the thesis(comprising of chapters 2,3,4 and 5) involves structural studies on the following features of protein-protein interactions: structural change, flexibility, symmetry, and residue conservation. The second part of the thesis(comprising of chapters 6,7,8 and 9) involves study of evolutionary aspects of protein-protein interactions, based on both large-scale data as well as case studies. Chapter1 provides a background and literature survey of the area of protein-protein interactions. The different classification schemes commonly used for describing the various protein-protein interactions are outlined. The key small-scale and large-scale experimental methods used for the identification of protein-protein interactions are described along with the details of the databases storing such experimentally derived information. Further, a comprehensive account of structural and evolutionary studies performed so far using the available data is provided. The computational(prediction)methods developed to address various aspects of protein-protein interactions are also outlined. In addition, the importance of protein-protein interactions in the context of diseases and the development of methods used to inhibit these interactions are discussed. Finally, the efforts towards design of protein-protein interactions based on the understanding of the principles governing their formation are outlined. Chapter 2 and chapter 3 describe different aspects of transient protein-protein interactions, which form an important subset of interactions, and are mainly involved in the regulation of Cellular processes. In chapter 2, the structural changes occurring upon complex formation are described. In chapter 3, roles of interface residues in the unbound form are described. In chapter 2, the nature, extent and location of structural changes upon binding is analyzed using a non-redundant and curated dataset of 77 structures of protein-protein complexes available in both bound and unbound forms. Structural change has been captured using two metrics: protein blocks and root mean square deviation of Cα positions. The relevance of the structural changes observed in protein-protein complexes is established by comparison with a control dataset of proteins not bound to any small or macromolecule. Results indicate that the observed changes are much larger than those observed due to random fluctuations. Given this background, the following observations were made on the nature, extent and location of structural changes in protein-protein complexes.(i) The nature of structural changes occurring at the interface is largely conformational with few rigid-body movements.(ii)The interfaces in the dataset are segregated into three types based on the extent of structural changes at the interface. A significant fraction of the interfaces are ‘pre-made’(almost in variant interface) or‘ induced-fit’(interface with large structural changes), while the rest are interfaces with moderate structural changes(‘others’). Analysis of structural changes using protein blocks reveals that pre-made interfaces are not completely invariant and are characterized by conformational changes of small magnitude. Pre-made interfaces are also observed to bind preferentially to‘ induced fit ’or‘ other’ interfaces. These observations implicate that non-obligate interactions possess in-built regulatory mechanisms in terms of conformational features to control the timely association-dissociation of transiently interacting proteins. (iii) Interestingly, significant structural changes away from the interface were observed in almost one-half of the complexes in the dataset. The analysis of these changes forms a major focus of this chapter. Crystallographic temperature factors, crystal packing, and normal mode analysis of these regions were studied to analyze the structural changes in these regions. Normal mode analysis along with literature survey indicates that most of the structural changes observed in non-interacting surface regions may be functionally relevant, with many of them corresponding to allosteric transitions. The majority of these changes occur in signaling proteins. The chapter summarizes that these observations suggest a much higher prevalence of allostery caused due to protein binding than appreciated before. The data set generated in this chapter can serve as a starting point to uncover potentially new allosteric modulators in signaling systems. In chapter 3, the question‘ Do residues at the interface of transient protein-protein interactions have any role in the unbound form?’ has been investigated. A high resolution, non-redundant and functionally diverse dataset of 67 proteins with known structures available both in the form of protein-protein complex and unbound forms has been used. Significantly low B-factors at the core of the interface in the unbound form are observed in these structures, indicating high rigidity. Many of these residues also show B-factors comparable to those of buried residues in a protein, which formed the basis for classifying interface residues as ‘rigid’ and‘ non-rigid’. These two types of residues have differential contribution towards the energetics of complex formation. It is also observed that rigid interfacial residues are conserved better in evolution than non-rigid interfacial residues. Their stronger selection is highlighted by substantial conservation of microenvironment (rigidness), sequence(amino acid identity/similarity) and structure(specific side-chain orientation) in homologous proteins. These observations coupled with the absence of any specific type of amino acid to occur preferentially at a rigid site indicates that rigidness is a property of the topological location of the residue at the interface and not the type of the amino acid present at that site. Analysis of the energetic parameters of these residues indicates that the y contribute significantly to the molecular recognition process by reducing the entropic cost on complexation by virtue of their pre-ordered conformation. This chapter also explores the contribution of interface residues towards the stability of the self-protein vis-à-vis that of the complex. It was seen that most interface residues contribute towards stabilizing the bound form. Interestingly, some of the interfacial residues predominantly stabilize the self-protein(the protein in which they are situated) and have a negligible contribution towards stabilization of the bound form. Thus, though these residues are located in the protein-protein interface their main role seems to be in the stabilization of the self-protein both in the unbound and bound forms. These residues are classified as Self-protein Stabilizing Residues(SSR -6.93%) and the rest as Neutrally Stabilizing Residues(NR -42.60%) and Complex Stabilizing Residues(CSR -50.46%). In addition, it was noted that the proportion of rigid residues is more in SSR(73.33%) than in NR(58.13%) and CSR(48.90%)sites. Apart from the favorable energetic contribution by rigid residues to the free energy of the unbound form than non-rigid residues, their predominance in SSRs suggests that rigid residues play an important role in the stabilization of the unbound form of the protein.The analyses performed in this chapter suggest that not all the protein-protein interfacial residues have the major role of stabilizing the complex; some of these residues seem to have more significant role in the unbound form than the bound form. Chapter4 provides a discussion on the prevalence and relevance of a symmetry in homodimeric proteins. One of the main features characterizing homodimers is the symmetric arrangement of subunits in the three-dimensional structures.Typically, asymmetric arrangements of subunits are associated with disease states; however, they are also observed in normal homodimers performing specialized functions. Two measures to quantify structural asymmetry in homodimers (global asymmetry and interface asymmetry)have been used on an on-redundant dataset of 223 biologically relevant homodimers. The survey for globally asymmetric homodimers in the dataset indicates that they are very rare(n=11).The chapter discusses cases where a globally asymmetric arrangement of homodimeric proteins has been utilized by the nature to perform certain specialized functions, such as linking of a dimeric system with a monomeric system(half-of-sites reactivity) and the transmission of signals emanating from asymmetric DNA repeats. Analysis of the 3-D structures of homologues reveals that there is no clear conservation of asymmetry. Specifically, the function of the homologous protein appears to dictate the pattern of structural organization. This chapter also describes the structural and evolutionary analyses of the 11 globally asymmetric complexes, which suggest possible mechanisms adopted by nature for preventing infinite array formation. The postulated mechanisms are:(i) In case of homodimers associating via non-topologically equivalent surfaces in their tertiary structures, ligand-dependent mechanisms are used.(ii) In case of homodimers associating via partially topologically equivalent surfaces, steric hindrance serves as the preventive mechanism of infinite array. Since most of the biologically relevant homodimers exhibit gross structural symmetry, this chapter explores further the extent of interface asymmetry in symmetric homodimers. It was observed that homodimers exhibiting grossly symmetric organization rarely exhibit either perfect local symmetry or high local asymmetry. Further, binding of small ligands at the interface does not cause any significant variation in interface asymmetry.The chapter provides new insights regarding accommodation of structural asymmetry in homodimers. Chapter 5 describes the ability of residue conservation of interface residues vis-à-vis surface residues near interface residues to identify fitting errors caused due to mis-orientation in cryo-electron microscopy maps. Cryo-electron microscopy is the most popular technique for solving structures of large assemblies in physiological conditions. However, the structures are usually solved at low resolution and atomic resolution is desired to get insights at the molecular-level. Although several methods have been developed for the fitting of atomic structures or models in to low-resolution cryo-electron microscopic maps, inaccurate fitting is observed in several cases. Using a non-redundant and high-resolution dataset of 125 permanent interactions and 95 transient interactions, it was observed that interface residues are significantly conserved better than residues near to the interface. The chapter describes the ability of this differential conservation to identify probable mis-fittings in cryo-EM maps for three case-studies: ribosomal complex from Escherichia coli, transferring-transferrin receptor complex from Homosapiens, and glutamate synthase complex from Azospirillum brasilense. For these cases, the use of conservation information resulted in the identification of a few residues in the vicinity of the interface with significantly higher conservation, implying their probable occurrence in the interface. These findings were verified against the high-resolution structures for two of these complexes (ribosomal assembly and transferring-transferrin receptor complex).These analyses suggest that residue conservation information can be useful in the fitting process to arrive at the fitted structure with an improved accuracy. Further, the discriminative power of the simplistic measure of residue conservation coupled with residue surface accessibility in identifying interacting residues on protein structures is also analyzed in this chapter. Testing on a set of signaling and scaffolding molecules indicates that this simplistic measure can identify interface residues in protein structures, indicating that conservation contains a distinct, although weak, signal for functional regions. Chapters 6 to 9 discuss studies involving evolutionary aspects of protein-protein interactions. Chapter 6 describes the usage of phylogenetic tree construction using maximum likelihood method to understand the origin of the signal captured by the mirror tree approach. Mirror tree is one of the most popular approaches for identifying interacting proteins based on co-evolution. This method uses the similarity in phylogenetic trees as an indicator of protein-protein interaction. The origin of the evolutionary signal detected by the mirror tree method is a subject of some controversy. Two broad hypotheses have been postulated in the literature to explain the origin of the signal(i)site-specific co-evolution alone and(ii)correlation induced by external factors with only minor, if any, contribution from site-specific co-evolution. In the typical mirror tree protocol, inferences from phylogenetic tree are optional and only genetic distances are analysed. Even if the tree is constructed, usually the Neighbor-Joining approach is used. However ,with Neighbor-Joining method the inferred tree topology and genetic distances are directly linked. With maximum likelihood the tree topology is not derived directly from the genetic distances and therefore the contributions of the signals arising from tree topology and genetic distances can be studied separately. Tree topologies can be considered to serve as indicators of compensatory substitutions(implicated in site-specific co-evolution)as well as shared evolutionary history. Genetic distances correspond to evolutionary rates(implicated in correlation induced by external factors).Using this method, phylogenetic trees for a range of datasets of interacting and non-interacting proteins corresponding to yeast(S.cerevisiae) have been derived. The analysis performed in this chapter reveals no strong correlation between phylogenetic tree topologies, and significant correlation between genetic distance matrices for interacting proteins. The chapter discusses the implications of these findings and attempts to understand the origin of the signal captured by mirror tree protocol using the following points.(i) The near lack of correlation in tree topologies is not surprising since compensatory substitutions accounts for only a minority of the sites in a protein.(ii) The influence of shared evolutionary history has also been tested in the chapter by comparison of tree topologies of interacting proteins and non-interacting with 18S rRNA tree. Tree topologies of both interacting and non-interacting proteins do not mirror the 18S rRNA tree, ruling out shared evolutionary history as the signal of correlated evolution.(iii) By contrast, the significant correlation observed between branch lengths(genetic distances) of interacting proteins in all the variant datasets demonstrates correlation between evolutionary rates, independent of evolutionary divergence. In summary, the chapter concludes by providing support for the theory of correlation induced by external factors with only minor contribution from site-specific co-evolution. Chapter 7 explores the homology based transfer of interactions by quantifying the extent of retention/variation of interaction partnerships amongst a set of homologous proteins related at SCOP family level(which indicates clear evolutionary relationship).A large dataset of domain-domain interacting pairs(n=20,254)culled from SCOP1.73 was used for this analysis. Study involving this dataset shows that in around~80% of the cases, interacting partners are completely retained(evaluated as proteins belonging to the same SCOP family).If‘common’ partnership is evaluated at the level of SCOP folds, which are known to be structurally similaral though not necessarily evolutionarily related, the percentage of homologous domains with complete retention of partnership increases only by~5%. This indicates that only the presence of a common structural scaffold is not a sufficient feature for interaction. Further, the chapter also describes the retention/variation in partnerships analyzed as a function of sequence divergence between the homologous proteins. It is observed that there is a higher tendency to vary interacting partners as the evolutionary divergence between the homologues increases. In spite of this, interaction partnerships appear to be retained for homologous domains irrespective of their sequence divergence if the function mandates the presence of the interaction. However, all these observations could be influenced by the incomplete nature of information on the interactions available in the structural space. This problem has been addressed in this chapter by studying variation in interaction partnerships for Saccharomyces cerevisiae proteins. Yeast was chosen since it is extensively studied and interactions are available for~87% of proteins yielding a comprehensive list of interactions. To study this aspect, the SCOP dataset of interacting proteins(which represents a generic dataset) was compared with interactions of homologous proteins from yeast. The dataset of interacting proteins for yeast collated from all sources and documented in BIOGRID v50 was used. In this analysis, the proportion of homologous domains showing complete retention of interacting partners was only ~12%. This observation is the reverse of the trend observed for the dataset of homologous SCOP domains. Further analysis of homologous pairs of yeast-SCOP domains, containing only those pairs whose interacting protein families are found both in yeast and SCOP dataset, was performed to ascertain the extent of contribution of organism-specific proteins to the variation in interaction partnership for homologous domains. The proportion of homologous domains showing complete retention of interaction partners increases to~50% for these cases. These observations indicate that organism-specific proteins contribute significantly to the variation of interaction partnerships in homologous proteins. The next two chapters(8 and 9) discuss two contrasting scenarios of interaction partnerships. Chapter 8 describes the study of two protein families showing variation in interaction partnerships/interface structure and analyzes the drift in protein-protein interaction surfaces in each of the cases. The analysis in this chapter is facilitated by the large number of sequences available for the case studies. The first case study involves members of the glutamine amido transferase (GAT) superfamily of enzymes. Three remote homologues in this superfamily could also be related by sequence: intracellular protease(DJ-1/PfpIfamily),C-terminal domain of the small subunit of carbomoyl phosphate synthetase (ClassI glutamine amidotransferase-like family), and C-terminal domain of catalase (Catalase ,C-terminal domain family).In two cases, it is seen that domain recruitment influences the interacting surface(catalase, carbamoyl phosphate synthetase). The tethered domains, which are involved in interaction with the GAT domain, are from different SCOP folds, indicating that partnerships are not retained at extreme divergence. However, members of the DJ-1/PfpIfamily form homodimers with differing quaternary structures i.e. different orientations of the dimers. Four members have been studied in detail in this chapter (intracellular protease–two distinct interfaces–forming hexamer, stress-induced protein -dimer, DJ-1protein -dimer, sigma cross-reacting protein -dimer). Since the members are sequentially less divergent(as they are within the same family), it is possible to trace the drift in interfaces among these members based on the multiple sequence alignments of members with the differing quaternary structures and the sequences bridging them. The second case study involves analysis of the family of legume lectins, which corresponds to another set of proteins exhibiting differing quaternary structures for remarkably well conserved tertiary structures and sequences. Analysis of variations in protein-protein interaction surfaces when they show only slight differences between homologous members indicates that the drift is gradual, as seen when tracing the dynamics of DJ-1 family members and legume lectin family members. There exist sequences containing many different intermediate combinations of the interacting residues involved in both the sets of proteins. Comparisons of homologues where an entire interface seems to be lost show a different trend(intracellular protease and DJ-1).The most prominent interacting residues show an abrupt shift between the two different subfamilies. However, inspection of the other interacting residues reveals that there is a gradual change occurring generally, although a drastic change in the important(although quantitatively smaller) residues would have led to loss of interface. In summary, analysis of the evolutionary dynamics of the consensus interface residues of different quaternary structure types of DJ-1/PfpI family of enzymes and legume lectins shows that nature employs only the most important mutations to Prevent a specific interface and form a new interface and the rest of the positions drift and accumulate changes in the course of evolution. Chapter 9 describes the opposite scenario i.e. conservation of an interface even at high sequence divergence, using the RNA polymerase assembly as a case study. The multi-molecular assembly consists of four core subunits–alpha (I and II), beta, betaprime, and omega. These four subunits are common to RNA polymerase complexes of eubacteria, eukaryota and archaea. The sigma subunit aids in initiation of transcription in eubacteria (cor eenzyme +sigma = holoenzyme). Remarkably, prokaryotic and eukaryotic structures exhibit high degree of structural similarity, although their sequence similarity is low(19-28% sequence identity).However, this is expected as the obligatory interaction between the various subunits is essential to successfully carry out transcription. This chapter investigates the structural accommodation of diverse sequences at the interface of RNA polymerase machinery of eubacteria, using sequence analysis and homology modelling. Analysis of domain composition and order of domains for the core subunits of the RNA polymerase assembly in>85 eubacterial species indicates complete conservation. However, conservation analysis of the various core subunits indicates that the interface residues are more divergent for alpha and omega subunits. Although beta and beta prime are generally well-conserved, the residues involved in interaction with the divergent subunits(i.e.alpha, omega) are not conserved. Insertions/deletions are also observed near the interacting surfaces even in the cases of most conserved subunits(beta and betaprime). The chapter describes the homology modeling of three divergent RNApolymerase complexes from Helicobacter pylori, Mycoplasma pulmonis and Onion yellows phytoplasma, highlighting that insertions/deletions can be accommodated near the interface as they generally occur at the periphery. The development of a generalized matrix capturing preferences of interface environment is documented, along with results comparing the similarity of the modeled interfaces to that of the template interface. It is observed that the modeled interfaces are physico-chemically similar to that of the template interfaces in Thermus thermophilus, indicating that nature accommodates substantial substitutions and insertions/deletions at and near the interface in order to retain the structure of the obligate complex, which is in dispensable for the process of transcription. The main conclusions of the entire thesis work are summarized in chapter10, which also places the work in the context of the field of protein-protein interactions. The new insights obtained for transient interactions and homodimers from structural studies are highlighted. The application of evolutionary conservation to improve fitting of atomic structures in cryo-electron microscopic maps is discussed. The understanding gained from study of different evolutionary aspects of protein-protein interactions, ranging from correlated evolution to evolutionary dynamics of variations in interactions is also highlighted. Appendix 1 of this thesis describes the homology modeling of the hexameric form of AAA ATPase domain of spastin along with associated structural analysis.

No description available.

published_or_final_version / abstract / Botany / Master / Master of Philosophy

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Human serum transferrin (hTF) is a bilobal glycoprotein that plays a central role in iron metabolism. Each lobe of hTF (N- and C-lobe) can reversibly bind a single ferric iron. Iron binds to hTF at neutral pH in the plasma; diferric hTF binds to specific hTF receptors (TFR) on the cell surface and the complex undergoes receptor mediated endocytosis. The pH within the endosome is lowered to ~5.6 and iron is released from hTF. Apo hTF remains bound to the TFR and recycles back to the cell surface. Upon fusion with the plasma membrane, apo hTF dissociates from the TFR and is free to bind more iron and continue the cycle. The iron release process is complicated by various factors which include pH, anions, a chelator, lobe-lobe cooperativity and interaction with the TFR. All of these influence iron release in a complex manner. Because they are intricately linked, it is difficult to determine the effect of any single parameter. We have utilized stopped-flow and steady-state fluorescence and urea gel electrophoresis to dissect the iron release process as a function of lobe-lobe interactions, the presence of the TFR, and changes in pH and salt concentration. Application of recombinant protein production and site-directed mutagenesis has allowed us to generate a variety of hTF constructs in which the iron status of each lobe is completely controlled. Thus, we have created authentic monoferric hTFs unable to bind iron in one lobe, diferric hTFs with iron locked in one lobe and diferric hTF in which iron can be removed from both lobes. Importantly, we have produced the soluble portion of the TFR (sTFR) to analyze interactions between hTF and the sTFR and to monitor iron release from hTF/sTFR complexes. Together, we are able to provide a more precise picture of iron release from the two lobes of hTF in the presence and absence of the TFR. Steady-state fluorescence emission scans and urea gel electrophoresis provide a qualitative evaluation of the iron status of each construct after a predetermined incubation in iron removal buffer (i.e. an endpoint). However, these techniques do not provide information regarding the kinetic pathway to reach that endpoint. Combined with stopped-flow fluorescence time-based kinetics, a more precise assessment of the iron release process has been obtained. We have determined that changes in pH and salt affect endpoint iron release from the C-lobe, but not the N-lobe, however, the kinetics of iron release from both lobes are highly sensitive to pH and salt. Kinetic analysis in the absence and presence of the sTFR reveals the complexity of the iron release process. In the absence of the sTFR, the kinetics of iron release are insensitive to the iron status of the opposite lobe. However, in the presence of the sTFR, the kinetics of iron release from both lobes are affected by the iron status of the opposite lobe. Determination of conformational changes induced by anion binding, lobe-lobe communication and sTFR interactions have now been confidently assigned. We have created kinetic models of iron release from diferric hTF ± the sTFR and incorporated specific events pertaining to anion binding, lobe-lobe communication and conformational changes associated with sTFR interactions. We provide irrefutable evidence that a critical role of the sTFR is to accelerate the rate of iron release from the C-lobe, while decreasing the rate of iron release from the N-lobe such that the two lobes effectively release iron on a time scale relevant to one cycle of endocytosis.

The thesis work contains two projects under the same umbrella. The first project is to provide a detailed analysis on the behavior of interfacial water molecules at protein-protein complexes, in this case focusing on homodimeric complexes, and to investigate their effect with respect to different residue types. For that reason the homodimeric data-set, which includes high-resolution (≤ 2.30 Å) X-ray crystal structures of 252 (140 Biological & 112 Non-biological) protein complexes was chosen to explore fundamental differences between interfaces that Nature has “engineered” vs. compared to interfaces found under man-made conditions. The data set was comprised of 5391 water molecules where a maximum of 4 Å from both interfacing proteins. Our analysis is applied a suite of modeling tools based on HINT, a program for hydropathic analysis developed in our laboratory. HINT is based on the experimental measurement of the hydrophobic effect. The second project is designed to explore various means of suppressing the expression of human genes that play critical role in HIV pathogenesis. To achieve this aim, a data set of Affymetrix Human HG Focus Target Array, which measures the expression levels of HIV seronegative and seropositive individuals in human PBMCs, was analyzed with Pathway Studio 9.0 software. This work gives insight into the elucidation of the important mechanisms of human proteins interactions in HIV seropositive individuals and their implications. Hence, we found the kind and types of microRNAs that are suppressing the human genes which have great role for HIV replication in a cell.

In this thesis we propose some new approaches to the study of complex networks, and apply them to multiple domains, focusing in particular on protein-protein interaction networks. We begin by examining the roles of individual proteins; specifically, the influential idea of 'date' and 'party' hubs. It was proposed that party hubs are local coordinators whereas date hubs are global connectors. We show that the observations underlying this proposal appear to have been largely illusory, and that topological properties of hubs do not in general correlate with interactor co-expression, thus undermining the primary basis for the categorisation. However, we find significant correlations between interaction centrality and the functional similarity of the interacting proteins, indicating that it might be useful to conceive of roles for protein-protein interactions, as opposed to individual proteins. The observation that examining just one or a few network properties can be misleading motivates us to attempt to develop a more holistic methodology for network investigation. A wide variety of diagnostics of network structure exist, but studies typically employ only small, largely arbitrarily selected subsets of these. Here we simultaneously investigate many networks using many diagnostics in a data-driven fashion, and demonstrate how this approach serves to organise both networks and diagnostics, as well as to relate network structure to functionally relevant characteristics in a variety of settings. These include finding fast estimators for the solution of hard graph problems, discovering evolutionarily significant aspects of metabolic networks, detecting structural constraints on particular network types, and constructing summary statistics for efficient model-fitting to networks. We use the last mentioned to suggest that duplication-divergence is a feasible mechanism for protein-protein interaction evolution, and that interactions may rewire faster in yeast than in larger genomes like human and fruit fly. Our results help to illuminate protein-protein interaction networks in multiple ways, as well as providing some insight into structure-function relationships in other types of networks. We believe the methodology outlined here can serve as a general-purpose, data-driven approach to aid in the understanding of networked systems.

The G-protein coupled receptors (GPCRs) are seven-transmembrane receptors that transduce signals from the cell surface to intracellular effectors. There are more than 1000 GPCRs in metazoans, while no GPCR has been definitively identified in plants. The most promising plant GPCR candidate, Arabidopsis G-protein coupled receptor 1 (GCR1), physically couples to the G-protein < subunit GPA1 and is involved in cell cycle regulation, blue light and phytohormone responses, but its signalling network remains largely unknown. This project aimed to achieve a better understanding of GCR1 signalling by identifying its interactors using a novel yeast two hybrid system – the Ras Recruitment System (RRS). Screening of an Arabidopsis cDNA library using a bait comprising intracellular loop 1 (i1) and 2 (i2) of GCR1 resulted in the isolation of 20 potential interactors. Extensive reconfirmation screening demonstrated that three of these interactors: Thioredoxin h3 (TRX3), Thioredoxin h4 (TRX4) and a DHHC type zinc finger family protein (zf-DHHC1) interact specifically with both i1 and i2 of GCR1. This was supported by the reverse RRS (rRRS) and 6xHis-pull-down assays. It is speculated that TRX3 and TRX4, which can reduce disulfide bridges of target proteins and act as powerful antioxidants, may regulate GCR1-mediated signalling events in response to oxidative stress. Alternatively, they may modulate GCR1 targeting or signalling through their chaperone activities. zf-DHHC1 has a predicted membrane topography that is shared by most DHHC domain-containing palmitoyl acyl transferases. It may modify GCR1 activity through palmitoylation of the two cysteines located at the cytoplasmic end of the first transmembrane domain. Together, these findings contribute to the growing understanding of the GCR1 signalling network, and provide valuable starting points for further investigation.

Le terme de sulfoprotéomique est utilisé pour désigner l’étude de la sulfatation des protéines. Bien que la sulfatation soit depuis peu considérée comme une MPT d’une importance majeure, il y a toujours peu de travaux scientifiques qui y sont consacrés en comparaison avec ce qui se fait sur la phosphorylation notamment. Ce retard s’explique notamment par la difficulté à analyser les espèces protéiques sulfatées dans les conditions classiques utilisées en protéomique, notamment par spectrométrie de masse. Ces travaux de thèse visent justement à développer des méthodes d’analyses par spectrométrie de masse dédiées à l’étude de la sulfatation des protéines, afin d’augmenter le champ des connaissances de cette MPT. Pour cela, nous avons largement utilisé le mode d’ionisation négatif, très peu, voire jamais utilisé en protéomique, avec deux techniques de fragmentation pour réaliser des spectres MS/MS, à savoir les fragmentations CID et HCD. Les résultats obtenus nous ont permis de mettre en évidence une méthode d’analyse permettant la formation d’ions spécifiques de la sulfatation et de la phosphorylation (qui sont isobariques), permettant ainsi une identification certaine de chacune des deux MPTs. Nous avons également entrepris d’étudier le rôle de la sulfatation d’un récepteur cellulaire, CXCR4, dans son interaction avec son ligand naturel, la chimiokine SDF-1/CXCL12. Cette étude a été menée par électrophorèse capillaire, et pourra constituer une base de travail solide pour des futures analyses mettant en œuvre le couplage entre l’électrophorèse capillaire et la spectrométrie de masse pour une meilleure caractérisation des complexes formés entre les partenaires protéiques. / Sulfoproteomics term designs protein sulfation studies. It appears during the 2000’s, when the interest for others Post-Translational Modifications (PTMs) than phosphorylation and glycosylation was growing up. Even though sulfation is thought to be an important PTM, a weak number of publications has emerged about it, notably if we compare with the huge quantity of phosphorylation papers. This difference is mainly due to the difficulty to correctly analyze sulfated proteins and peptides in the classical ways of proteomics, as in mass spectrometry for example. The goal of this thesis is to develop mass spectrometry methods dedicated to the characterization of sulfated species, in order to improve the knowledge of this PTM. To do that, we have mainly used negative ion mode, which is almost never used, with two fragmentations techniques for the MS/MS spectra, which are CID and HCD. Results obtained allow us to pinpoint an analytical method allowing the differentiation between sulfation and phosphorylation (they are isobaric), based on the presence of specific ion for each PTM in MS/MS. In another part of the project, we have investigated the role of sulfation in the interaction between a cellular receptor, CXCR4, and its in vivo ligand, the chemokine SDF-1/CXCL12. We used capillary electrophoresis for this work, and it could be a good basis for future analyses using capillary electrophoresis coupled with mass spectrometry, in order to have a better characterization of the observed complexes.

氫化酶作為一種催化劑，能催化氫分子成為質子及電子的相互轉換。 [鎳鐵]- 氫化酶散播最廣的一種氫化酶，從古菌到細菌都能找到 [鎳鐵]- 氫化酶。完整成熟的 [鎳鐵]-氫化酶需要插入鐵、氰化物、一氧化碳以及鎳到它的催化核心。這複雜的過程需要其它由若干 hyp 基因編譯的輔助蛋白酶的幫助，其中蛋白HypA 與 HypB 負責將鎳運送到[鎳鐵] -氫化酶的催化核心。敲除了 hypA 或hypB 基因的細菌株缺失[鎳鐵] -氫化酶的活性，如在生長介質裡添補鎳可恢復部份[鎳鐵] -氫化酶的活性。當HypB 與鳥嘌呤核苷酸結合時會變成蛋白二聚體。對比HypB 脫輔基蛋白及與HypB 與鳥嘌呤三核苷酸類似物的蛋白複合物的晶體結構可發現，HypB 透過一個保守賴氨酸殘基( Archaeoglobus fulgidus HypB 的殘基 148 )組成分子間鹽橋以構成蛋白二聚體。Escherichia coli 的體內實驗顯示，此保守賴氨酸殘基對活性氫化酶的製造起必要的作用，反映由此殘基所構成的鹽橋對HypB 功能的重要性。此外，本研究展示了A. fulgidusHypA 及 HypB 蛋白之間的相互作用。通過在A. fulgidus HypB 上進行系統性的突變，發現HypB 利用其GTP 酶域上的一段氨基端區域與HypA 相互作用。跟據這個結果，我們進而在E. coli HypB 上發現了兩個保守的非極性殘基與HypA 相互作用。當以丙氨酸取代在HypB 上的這兩個非極性殘基時，HypB 無法激活E. coli 中的氫化酶，導置降低的氫化酶活性，這表明了HypA 和HypB 的相互作用對[鎳鐵] -氫化酶成熟過程的必要性。 / Hydrogenases catalyze the inter-conversion of molecular hydrogen into protons and electrons. [NiFe]-hydrogenase is the most widely distributed hydrogenases, which is found in organisms ranging from archaea to bacteria. Maturation of [NiFe]-hydrogenase requires the insertion of iron, cyanide and carbon monoxide, followed by nickel, to the catalytic core of the enzyme. The maturation process of hydrogenase is a complicated procedure, which requires many accessory proteins encoded by hyp genes. HypA and HypB participate in the nickel delivery step to the catalytic core of hydrogenase, which is supported by the fact that strain deficient in hypA or hypB gene lack hydrogenase activity which can be recovered partially by elevating nickel content in the medium. HypB is capable to form dimer in solution upon guanine nucleotide binding. By comparing the crystal structures of HypB in dimer and monomer form, an important lysine residue (residue 148 in A. fulgidus HypB) which is required to form an intermolecular salt bridge during GTP-dependent dimerization, has been identified. Substitution of this lysine resiue with alanie would break HypB dimer in vitro. In vivo complementation study in E. coli showed that the corresponding lysine residue in E. coli HypB is required for active hydrogenase production indicating the importance of this intermolecular salt bridge to the biological function of HypB. Besides, interaction between A. fulgidus HypA and HypB are demonstrated in this work. By making systematic mutation to A. fulgidus HypB, the N‐terminal region of the GTPase‐domain has been identified to be important for its interaction with HypA. Further mutagenesis study has been done on E. coli HypB and two conserved non‐polar residues responsible for interaction with HypA have been identified. Alanine substitution of these conserved non‐polar residues result in HypB mutants which failed to rescue hydrogenase activity in vivo in E. coli showing that HypA/HypB interaction is required for hydrogenase maturation. / Detailed summary in vernacular field only. / Chan, Kwok Ho. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 88-95). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Chapter Chapter 1 --- Introduction: Hydrogenase biosynthesis requires insertion of nickel facilitated by protein HypA and HypB --- p.1 / Chapter 1.1 --- What is hydrogenase? --- p.1 / Chapter 1.2 --- [NiFe] hydrogenase contains a complex catalytic core composed of metal atoms and diatomic ligands --- p.2 / Chapter 1.3 --- The [NiFe] catalytic core --- p.4 / Chapter 1.4 --- Building the catalytic [NiFe] core --- p.4 / Chapter 1.5 --- Nickel insertion into the hydrogenase precursor involves the proteins HypB, HypA and SlyD --- p.7 / Chapter 1.5.1 --- Protein HypB --- p.7 / Chapter 1.5.2 --- Protein HypA --- p.11 / Chapter 1.5.3 --- Protein SlyD --- p.12 / Chapter 1.6 --- Objectives - How HypB dimerization and HypA/HypB interaction are involved in hydrogenase maturation process? --- p.13 / Chapter Chapter 2 --- A conserved Lys residue is required for GTP-dependent dimerization and hydrogenase maturation --- p.17 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Materials and Methods --- p.22 / Chapter 2.2.1 --- Recombinant Plasmid Construction --- p.22 / Chapter 2.2.2 --- HypB mutant construction by site-directed Mutagenesis --- p.22 / Chapter 2.2.3 --- Protein Expression and purification --- p.23 / Chapter 2.2.4 --- HypB protein purification --- p.23 / Chapter 2.2.5 --- Analytical gel filtration chromatography coupled with Light Scattering (SEC/LS) --- p.24 / Chapter 2.2.6 --- Nucleotide binding affinity determination --- p.25 / Chapter 2.2.7 --- GTPase activity determination --- p.26 / Chapter 2.2.8 --- Sample preparation for hydrogenase activity assay --- p.26 / Chapter 2.2.9 --- Hydrogenase activity determination --- p.27 / Chapter 2.3 --- Results --- p.29 / Chapter 2.3.1 --- AfHypB undergoes GTP-dependent dimerization --- p.29 / Chapter 2.3.2 --- Analysis of Structural difference between the apo form and GTP S-bound form suggests a mechanism of GTP-dependent dimerization for HypB --- p.30 / Chapter 2.3.3 --- Lys-148 is essential for GTP-dependent dimerization --- p.31 / Chapter 2.3.4 --- Disruption of dimerization by K148 mutation did not affect nucleotide binding and GTP hydrolysis activity significantly --- p.32 / Chapter 2.3.5 --- The conserved lysine residue is required for hydrogenase maturation in E. coli --- p.33 / Chapter 2.4 --- Discussion --- p.45 / Chapter 2.4.1 --- A conserved intermolecular salt‐bridge is required for GTP-dependent dimerization of HypB and hydrogenase maturation --- p.45 / Chapter 2.4.2 --- The extra metal binding site at the dimeric interface of HypB may provide a mechanism of why GTP-dependent dimerization is essential to Ni insertion --- p.46 / Chapter Chapter 3 --- N-terminal region of GTPase‐domain of HypB is required for interaction with HypA --- p.51 / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Methods and materials --- p.53 / Chapter 3.2.1 --- Recombinant Plasmid Construction --- p.53 / Chapter 3.2.2 --- HypB variant construction by site‐directed Mutagenesis --- p.53 / Chapter 3.2.3 --- Protein Expression --- p.54 / Chapter 3.2.4 --- Tag‐free AfHypA and AfHypB purification --- p.54 / Chapter 3.2.5 --- Analytical size exclusion chromatography coupled with Light Scattering --- p.54 / Chapter 3.2.6 --- GST pull‐down of GST‐AfHypA and AfHypB --- p.55 / Chapter 3.2.7 --- Tandem affinity pull‐down of GST‐EcHypA and His‐SUMO‐EcHypB --- p.55 / Chapter 3.2.8 --- GST pull‐down of GST‐EcHypA and His‐SUMO‐EcHypB --- p.56 / Chapter 3.2.9 --- Hydrogenase activity determination --- p.57 / Chapter 3.3 --- Results --- p.58 / Chapter 3.3.1 --- HypA and HypB from A. fulgidus form 1:1 heterodimer in solution --- p.58 / Chapter 3.3.2 --- The N‐terminal regions upstream of the first helix of A. fulgidus HypB is required for HypA-HypB interaction --- p.59 / Chapter 3.3.3 --- Two conserved hydrophobic residues on HypB from E. coli are required to interact with HypA --- p.60 / Chapter 3.3.4 --- HypA-HypB interaction is required for hydrogenase maturation in E. coli --- p.62 / Chapter 3.4 --- Discussion --- p.73 / Chapter 3.4.1 --- The N‐terminal region of the GTPase domain is required for interaction with HypA and hydrogenase maturation in E. coli --- p.73 / Chapter 3.4.2 --- Location of interaction site on HypB reveals possible role for HypA/HypB interaction --- p.74 / Chapter 3.4.3 --- Mode of specific interaction with HypA: Interaction via a disordered region implies a coupled folding and binding process --- p.75 / Chapter Chapter 4 --- Conclusion and Future Perspectives --- p.80 / Chapter A1.1 --- Summary of findings in this work --- p.80 / Chapter A1.2 --- Implications in hydrogenase maturation --- p.81 / Chapter A1.3 --- Questions unresolved --- p.82 / Chapter 4.3.1 --- Factors that activate GTPase activity of HypB are still elusive --- p.82 / Chapter 4.3.2 --- How nickel delivery is regulated by HypA/HypB complex is still unclear --- p.83 / References --- p.88 / Chapter Appendix 1 --- Preliminary results of HypA/HypB protein complex structural study --- p.96 / Chapter A1.1 --- Structural study may provide invaluable insights to the role of HypA‐HypB interaction --- p.96 / Chapter A1.2 --- X‐ray crystallography as an approach to determine HypA/HypB complex structure --- p.96 / Chapter A1.3 --- Initial crystal hits were obtained with purified AfHypA/HypB complex --- p.97 / Chapter Appendix 2 --- Publications associated to the thesis --- p.100 / Chapter Appendix 3 --- Constructs and Primers used --- p.101