Dissertations / Theses on the topic 'Periplasmic phosphate binding protein'

La surproduction par escherichia coli de la proteine periplasmique affine pour le phosphate (phos) provoque l'accumulation du precurseur de phos a la fois dans la membrane interne et le cytoplasme. Seul le precurseur membranaire peut etre mature et exporte, alors que le precurseur cytoplasmique subit une coupure lente de sa sequence signal et donne naissance a une forme "pseudo-mature" cytoplasmique. Existence d'un captage entre la synthese et l'exportation. Une application du systeme phos a ete realisee pour la production du facteur de liberation de l'hormone de croissance humaine (hgrf) chez e. Coli. En carence de phosphate, la proteine hybride phos-hgrf est synthetisee et exportee vers le periplasme

Haemophilus ducreyi, a Gram-negative and heme-dependent bacterium, is the causative agent of chancroid, a genital ulcer sexually transmitted infection. Although the precise molecular mechanism of heme acquisition in H. ducreyi is unclear, heme uptake likely proceeds via a receptor mediated process. The initial event involves binding to either of two outer membrane receptors, TdhA and HgbA. Once heme is deposited into the periplasmic space, a heme permease is postulated to transport heme across the periplasmic space to the inner membrane. In prior experiments, using protein expression profiling of the H. ducreyi periplasmic proteome, we identified a periplasmic-binding protein hHBP that we propose is a component of a heme trafficking operon. Biochemical and genetic approaches were used to functionally characterize hHBP. First, purified hHBP was incubated with increasing concentrations of heme and the mixtures were resolved by non-denaturing polyacylamide gel electrophoresis. Separated proteins were transferred onto PVDF membranes and heme-protein complexes were detected by enhanced chemiluminescence (ECL). Second, the hhbp gene was cloned in the E. coli recombinant mutant E. coli FB827 dppA::Km mppA::Cm (pAM238-HasR) which expresses the Serratia marcescens HasR heme receptor allowing heme translocaton into the periplasm, but denies heme entry into the cytoplasm because of the presence of the double mutation (dppA::Km mppA::Cm) resulting in a mutant lacking the two periplasmic proteins DppA and MppA. We found that heme binding to hHBP was saturable as determined by ECL. Genetic complementation in trans with hhbp repaired the growth defect of the mutant E. coli for heme utilization as an iron source. The growth restoration was comparable to that seen with the E. coli mutant complemented with the intact Dpp permease. Additionally, growth of the mutant was not rescued with the empty plasmid vector. We concluded that H. ducreyi hHBP functionally binds heme. Complementation of the E. coli mutant for heme competency supports the proposal that hHBP participates in the transit of heme in H. ducreyi.

In Haemophilus ducreyi, heme uptake likely proceeds via a receptor-mediated process. The initial event involves binding to either of two outer membrane receptors, TdhA and HgbA. Once heme is deposited into the periplasmic space, we hypothesize that a heme-dedicated periplasmic binding protein (hHBP) is responsible for transporting heme across the periplasmic space to the inner membrane. To identify the hHBP, periplasmic extracts were generated from H. ducreyi 35000 grown under high and low heme conditions and subjected to proteome mapping. Peptide sequences of upregulated proteins grown under heme-restrictive conditions were determined by mass spectroscopy. A candidate hHBP was identified as a periplasmic binding protein homologous to YfeA of Yersinia pestis. The gene encoding this protein appears to be in a typical ABC transporter operon. Under iron-limiting conditions, no upregulation of the hHBP expression was observed; however, the purified hHBP was shown to specifically bind heme in a concentration-dependent manner.

Les « phosphate binding protein » (PBP) constituent une famille de protéines présentes de manière ubiquitaire chez les bactéries et plus marginalement chez les Eucaryotes. Impliquées dans l'import du phosphate extracellulaire chez les bactéries, les PBPs présentent un site de fixation du phosphate très bien caractérisé avec, notamment, une liaison hydrogène particulière nommée «low barrier hydrogen bond» (LBHB). Cette LBHB est impliquée dans la discrimination entre le phosphate et des anions proches chez les PBPs. Bien que cette discrimination semble nécessiter une haute conservation du site de fixation du phosphate, dans la nature différentes configurations sont observées. Au cours de ce travail, nous nous sommes intéressés à la PBP d'un organisme pathogène, C.perfringens qui présente un site de fixation alternatif. Avec, entre autre, une perte de la LBHB, cette PBP présente la plus faible capacité de discrimination testée à ce jour. Cette faible capacité de discrimination pourrait être liée au biotope de la bactérie ou bien à un phénomène d'adaptation fonctionnelle. D'autre part, certaines PBPs présentent des propriétés d'inhibition du VIH via l'étape de la transcription virale. Cependant, ces protéines sont particulièrement difficiles à produire en système hétérologue limitant l'étude fonctionnelle. Afin de lever ce verrou technique, nous avons développé une nouvelle méthodologie basée sur la phylogénie en vue de solubiliser notre modèle d'étude (HPBP). Nous avons obtenu un variant soluble de HPBP qui conserve ses activités antivirales permettant de débloquer les études fonctionnelles
The "phosphate binding protein" constitutes a family of proteins ubiquitously found in Prokaryotes but also more sparsely distributed in Eukaryotes. Involved in phosphate import, PBPs exhibits a well-characterized phosphate binding site with a peculiar hydrogen bond called "low barrier hydrogen bond" (LBHB). This LBHB is involved in the unique discrimination properties of PBPs, capable of discriminating phosphate from other similar anions such as arsenate of sulfate. Albeit this high discriminating property needs a high conservation of the phosphate binding pocket, different configurations are observed in nature. Herein, we have been interested in a PBP from a human pathogen, Clostridium perfringens, which presents an alternative phosphate binding site. Exhibiting a loss of the LBHB, C.perfringens PBP is the least discriminating PBP isolated so far. This weak discrimination property might be related to the environment of C.perfringens or to a functional adaptation of the PBP. On the other hand, PBPs issued from eukaryotic tissues exhibit HIV inhibition properties via a step not yet targeted in current therapies, i.e. the transcription. However, these proteins are difficult to obtain from human tissues and their expression in heterologous system remains impossible. We have developed a new methodology based on phylogeny in order to solubilise our study model, HPBP. Thus, we have obtained a soluble variant of HPBP which conserves the HIV-inhibiting properties. This unique tool both allow to unlock functional studies and lead to a better understanding on how PBPs are capable of inhibiting HIV

Biometals such as copper, cobalt and zinc are essential to life. These transition metals are used as cofactors in many enzymes. Nonetheless, these metals cause deleterious effects if their intracellular concentration exceeds the cells' requirement. Prokaryotic organisms usually employ efflux systems to maintain metals in appropriate intracellular concentrations.The Cus system of Escherichia coli plays a crucial part in the copper homeostasis of the organism. This system is a tetrapartite efflux system, which includes an additional component compared to similar efflux systems. This fourth component is a small periplasmic protein, CusF. CusF is essential for full copper resistance, yet its role within the Cus system has not been characterized. It could potentially serve in the role of a metallochaperone or as a regulator to the Cus system.To gain insight into the molecular mechanism of resistance of this system, I have structurally and biochemically characterized CusF. Using X-ray crystallography I determined the CusF structure. CusF displays a novel fold for a copper binding protein. Through multiple sequence alignment and NMR chemical shift experiments, I proposed a metal binding site in CusF, which I confirmed through determination of the structure of CusF-Ag(I). CusF displays a novel coordination of Ag(I) and Cu(I) through a Met2His motif and a cation-pi interaction between the metal ion and a tryptophan sidechain. Furthermore, I have shown that CusF binds Cu(I) and Ag(I) specifically and tightly.I investigated the role of the tryptophan at the binding site to establish its effect on metal binding and function of CusF. I have shown through competitive binding assays, NMR studies and through collaborative EXAFS studies that the tryptophan plays an essential role in CusF metal handling. The affinity of CusF for Cu(I) is influenced by this residue. Moreover, the tryptophan also caps the binding site such that oxidation of the bound metal as well access to adventitious ligands is prevented. In summary, these findings show that the structure and metal site of CusF are unique and are specifically designed to perform the function of CusF as a metallochaperone to the Cus system.

Nicotinic acid adenine dinucleotide phosphate (NAADP) has recently emerged as a novel intracellular calcium mobilising messenger in a variety of cells. Whilst increasing evidence suggests that NAADP acts on a distinct binding protein, little is known regarding the biochemical properties of the putative NAADP "receptor". My thesis investigates properties of the NAADP-binding protein in sea urchin eggs. Firstly, I show that NAADP binding to its target protein is inhibited by altering the protein:lipid ratio of soluble sea urchin egg homogenates - an effect prevented and reversed specifically by addition of exogenous phospholipids. These data highlight the importance of the lipid environment in maintenance of NAADP binding to its target protein. In addition, I show that upon binding its ligand, the NAADP-binding protein undergoes an unusual stabilization process that is dependent upon the time the receptor is exposed to its ligand. This property endows the NAADP-binding protein with the extraordinary ability to detect the duration of its activation. Finally I describe the development of a highly sensitive radioreceptor assay (based upon the sea urchin egg NAADP-binding protein) that is capable of detecting low levels of NAADP from cellular extracts. I apply this technique to determine NAADP levels in a variety of extracts prepared from cells under resting and stimulated conditions.

Haemophilus influenzae is a pathogenic Gram-negative bacterium that colonizes the upper respiratory tract of humans and can cause otitis media, upper and lower respiratory infections, and meningitis. Factors important for H. influenzae to colonize humans and cause disease are not fully understood. Different bacterial pathogens are armed with virulence mechanisms unique to their specific strategies for interacting with their hosts. Many of the proteins mediating these interactions are secreted and contain disulfide bonds required for function or stability. I postulated that identifying the set of secreted proteins in H. influenzae that require periplasmic disulfide bonds would provide better understanding of this bacterium's pathogenic mechanisms. In this thesis, the periplasmic disulfide bond oxidoreductase protein, DsbA, was found to be essential for colonization and virulence of H. influenzae. Mutants of dsbA were also found to be sensitive to the bactericidal effects of serum. However, the DsbA-dependent proteins important for pathogenesis of this organism have not been previously identified. To find them, putative targets of the periplasmic disulfide bond pathway were identified and examined for factors which might be important for mediating critical virulence aspects. By doing so, novel virulence factors were discovered including those important for heme and zinc acquisition, as well as resistance to complement. Overall, the work presented here provides insight into requirements for H. influenzae to survive within various host environments.

Les bactéries Escherichia coli et Shewanella oneidensis sont capables d'utiliser l'oxyde de triméthylamine (TMAO) comme accepteur terminal d'électrons pour leur respiration. La réduction du TMAO est réalisée par le système respiratoire TMAO réuctase codé par l'opéron tor. Chez ces deux bactéries, l'expression de cet opéron est sous le contrôle strict du TMAO et sa régulation fait intervenir un système à deux composants TorS/TorR et une protéine périplasmique TorT. Le premier objectif de ma thèse a été de déterminer comment le TMAO était détecté par le système de régulation à deux composants TorS/TorR. Il s'avère que les protéines TorT d'E. Coli et de S. Oneidensis sont des protéines affines du TMAO. En présence de TMAO, la protéine TorT interagit avec le senseur TorS qui est alors capable de phosphoryser son régulatuer de réponse partenaire TorR. En absence de TMAO, la protéine TorT de S. Oneidensis est libre dans le périplasme tansi que celle d'E. Coli interagit toujours avec TorS. J'ai montré que, chez E. Coli, la présence de TMAO induit une cascade de changements conformationnels de TorT à TorS permettant ainsi la transduction du sinal TMAO. Des homologues de la protéines TorT ont été indentifiés et leurs gènes peuvent être classés en deux catégories selon leur localisation au sein du locus tor ou à proximité de gènes codant pour un ABC transformateur putatif. Un des produits de cette deuxième catégorie étant également capable de fixer le TMAO, il pourrait donc être impliqué dans l'import du TMAO. Nous proposons donc l'existence d'une nouvelle famille de protéines affines du TMAO ou de composés apparentés impliquée dans la transduction du signal ou dans le transport. Le deuxième objectif de ma thèse a été d'étuder le comportement chimiotactique de S. Oneidensis vis-à-vis du TMAO. Nous avons montré que le chimiotactique vis-à-vis du TMAO, ainsi que vers d'autres accepteurs d'électrons comme le nitrite, le nitrate, le fumarate ou le DMSO, résulte d'un mécanisme d'énergie taxie. Dans ce mécanisme, la fonction des oxydoréductases est requise et la force proton motrice générée lors de la respiration constitue le signal. Le chimiorécepteur principalement impliqué dans le mécanisme d'energie taxie a également été identifié. Ce chimiorécepteur possédant un motif Cache, nous proposons que ce domaine soit impliqué dans la détection de la force proton motrice. En conclusion, chez S. Oneidensis, le TMAO constitue le signal qui induit l'expression du système Tor dont le fonctionnement induit à son tour une réponse chimiotactique de type énergie taxie vis-à-vis du TMAO
Escherichia coli and Schewanella oneidensis ca use trimethylamine N-oxide (TMAO) as terminal electron acceptor for respiration. Reduction of TMAO in TMA (trimethylamine) is mainly performed by th TMAO reductase system encoded by the tor operon. The expression of this operon is under the strict control of TMAO. This regulation involves the TorS/TorR two-component system and the TorT periplasmic protein. The first goal of mys thesis was to determine how the TorS/TorR two-component system detects TMAO. It turns out that TorT of E. Coli and of S. Oneidensis are TMAO-binding proteins. In the presence of TMAO, TorT interacts with the sensor TorS wich is then able to phosphorylate its cognate response regulator TorR. In the absence of TMAO, the TorT protein of S. Oneidensis is free in the periplasm whereas the TorT protein of E. Coli still interacts with TorS. I have shown that, in E. Coli, the presence of TMAO induces a cascade of conformational changes from TorT to TorS, leading to TorS activation. Several homologues or TorT were identified and their genes are located either in tor loci or closed to genes coding for putative ABC transporters. One member of this second class also binds TMAO and could thus be involved in the import of TMAO. We proposed that TorT homologues constitue a new family of periplasmic binding proteins dedicated to signal detection or transport of TMAO or related compounds. The second goal of my thesis was to study the chenomatic behaviour of the S. Oneidensis towards TMAO. I have shown that taxis towards TMAO and other exgenous electron acceptors, including fumarate, nitrate and DMSO, is governed by an energy taxis mechanism. The activity of terminal oxydoreductases is required and the proton motive force generated by respirationprovides the signal. The dedicated chemoreceptor was identified, and it contains a Cache domain. We proposed that this domain mediates the detection of the proton motive force. To conclude, in S. Oneidensis, TMAO is the signal that induces the expression of the Tor system, whose acivity induces, in turn, a chemotactic response towards TMAO

The analysis of gene changes associated with formation of the mycorrhizal symbiosis between orchid and fungi could have broad implications for plant pathogen interactions. Fungi associated with North American terrestrial orchids were once included in the pathogenic genus Rhizoctonia. This suggests that orchids are able to overcome or utilize normally pathogenic pathways to establish symbioses. A differential display technique was employed to analyze gene changes in orchid in response to a fungus. Samples of RNA from roots of Cypripedium parviflorum var. pubescens (CyPP) grown in the presence or absence of a mycorrhizal fungus; Thanatephorus pennatus, were analyzed using AFLP differential display. Forty-four fragments were selected out of 5000 as being differentially expressed, but only 15 sequences were obtained. Most showed homology to ribosomal genes. Two represented genes believed to be regulated by the mycorrhizal interaction: trehalose-6-phosphate synthase/phosphatase (Tps), which showed down-regulation and nucleotide binding protein (NuBP), which showed up-regulation. The Tps partial clone identifies 2100 bp at the 3' end of the gene and encodes a protein of 667 amino acids. The NuBP gene is approximately 1200bp in length and encodes a protein of 352 amino acids. The Tps gene exists in multiple copies with high expression in roots and low expression in rhizomes and leaves. The NuBP gene exists as a single copy and has a low level of expression in rhizomes and leaves. Expression of Tps is induced by sucrose, but reduced by trehalose. Cultivation of CyPP with non-mycorrhizal fungi did not affect expression of Tps or NuBP. Trehalose induced NuBP expression whereas sucrose did not. A second species of mycorrhizal fungi induced expression of NuBP but reduced expression of Tps. Analysis of Tps expression in Arabidopsis was done using promoter:GUS fusions. The Tps promoter:GUS plants revealed that Tps expression is constitutive in roots. Regulation of Tps driven GUS is expressed throughout seedlings. GUS was not detected in leaves of older plants but was detected in anthers and stigmatic surfaces of flowers. Expression of GUS driven by Tps showed a strong wound response and was present in the junction between siliques and pedicels.
Ph. D.

This dissertation is dedicated to the research and investigation of novel enzymes and the methods used to study them, with physiological roles ranging from isoprenoid biosynthesis to neurotransmitter production. Using a combination of bioinformatics, recombinant cloning, enzymology, and proteomics, we have contributed to the understanding and exploration of several human illnesses, including malaria, cancer, and endocrine dysfunction. Our first project involved studying the enzymes responsible for N-acylarylalkylamide biosynthesis in Bombyx mori. Very little is known how these potent signaling molecules are produced in vivo, however, one possible pathway is the direct conjugation of an acyl-CoA to a corresponding arylalkylamide by the enzyme arylalkylamine N-acyltransferase (AANAT). In insects, this enzyme is responsible for controlling melanism, the inactivation of biogenic amines, the sclerotization of the insect cuticle, photoperiodism, and the penultimate intermediate in the production of melatonin. We studied a pair of AANAT enzymes from B. mori: Bm-AANAT and Bm-AANAT3. The former was found to catalyze the direct formation of long-chain acylarylalkylamines (only the second enzyme discovered to do such chemistry), while the latter exhibited potent inactivation of several amines through acetylation. We conducted a full kinetic characterization of Bm-AANAT3, including double-reciprocal plots, pH-rate profiling, dead-end inhibition, and the construction of mutants to elucidate catalytically-relevant amino acids. Our hope is that new insights and discoveries on these enzymatic pathways in model organisms will yield novel molecular targets for human health and disease. We then developed an innovative, microwave-assisted synthesis of a binding-based probe capable of enriching proteins that bind adenine ribose derivatives (AdoRs). We employed this probe in activity-based protein profiling studies to qualitatively assess the AdoR-binding proteome in three bacterial cell lines from the genus Bacillus. This proof of concept experiment demonstrated a unique subset of proteins distinctive to each species, and confirmed the efficacy of the probe tagging and subsequent enrichment. This technology can be used in clinical applications for the detection and identification of cancerous biomarkers. Finally, we successfully truncated and recombinantly-expressed 1-deoxy-D-xylulose-5-phosphate synthase (DXS) from both P. vivax and P. falciparum. We elucidated the order of substrate binding for both of these TPP-dependent enzymes using steady-state kinetic analyses, dead-end inhibition, and intrinsic tryptophan fluorescence titrations. Both enzymes adhere to a random sequential mechanism with respect to binding of both substrates: pyruvate and D-glyceraldehyde-3-phosphate. These findings are in contrast to other TPP-dependent enzymes, which exhibit classical ordered and/or ping-pong kinetic mechanisms. A better understanding of the kinetic mechanism for these two Plasmodial enzymes could aid in the development of novel DXS-specific inhibitors that might prove useful in treatment of malaria.

Dans la plupart des cellules eucaryotes, l'inositol hexakisphosphate (insp#6, acide phytique) est le phosphoinositol le plus abondant. Chez l'amibe dictyostelium discoideum, la concentration d'insp#6 est de 0,7 mm et se maintient lors du cycle de differenciation de l'amibe. La fonction intracellulaire de cet inositol polyphosphate n'est pas clairement elucidee, toutefois un role regulateur de l'insp#6 dans le processus d'endocytose a recepteurs a ete propose chez les mammiferes. Un test de liaison de l'insp#6 radioactif a ete tout d'abord mis au point afin de pouvoir suivre les proteines capables de lier ce phosphoinositide, lors de purifications a partir d'extraits solubles de d. Discoideum. En combinant cette methode avec un test immunologique permettant de detecter les chaines lourdes des adaptines (proteines impliquees dans l'endocytose a recepteurs), le protocole de purification a permis de mettre en evidence cette proteine chez l'amibe. L'adaptine chez d. Discoideum ne semble pas fixer l'insp#6 contrairement a la situation dans les cellules de mammiferes. Une purification des proteines cytosoliques de liaison de l'insp#6 a permis l'identification de trois proteines non decrites jusqu'a present chez l'amibe : une nouvelle isoforme de cyclophiline et les proteines ribosomales s4 et s10. Le clonage de leur gene respectif ainsi que l'etude de leur expression au cours du developpement de l'amibe ont ete entrepris. Les trois genes sont specifiques du stade vegetatif : leur niveau de transcription s'effondre apres le commencement de la differenciation. La cyclophiline b purifiee est capable de lier la ciclosporine a et possede une extension n-terminale qui est clivee apres traduction de la proteine. Cette sequence signal permet son adressage vers le reticulum endoplasmique : la cyclophiline peut se trouver dans ce compartiment ou dans une vesicule de la voie de secretion, en effet aucun signal de retention dans le reticulum n'a ete trouve dans sa sequence. Une autre isoforme de cyclophiline localisee dans la mitochondrie a ete obtenue par chromatographie par affinite pour la ciclosporine a.

Native states of globular proteins typically show stabilization in the range of 5 to 15 kcal/mol with respect to their unfolded states. There has been a considerable progress in the area of protein stability and folding in recent years, but increasing protein stability through rationally designed mutations has remained a challenging task. Current ability to predict protein structure from the amino acid sequence is also limited due to the lack of quantitative understanding of various factors that defines the single lowest energy fold or native state. The most important factors, which are considered primarily responsible for the structure and stability of the biological active form of proteins, are hydrophobic interactions, hydrogen bonding and electrostatic interactions such as salt bridges as well as packing interactions. Several studies have been carried out to decipher the importance of each these factors in protein stability and structure via rationally designed mutant proteins. The limited success of previous studies emphasizes the need for comprehensive studies on various aspect of protein stability. An integrated approach involving thermodynamic and structural analysis of a protein is very useful in understanding this particular phenomenon. This approach is very useful in relating the thermodynamic stability with the structure of a protein. A survey of the current literature on thermodynamic stability of protein indicates that the majority of the model proteins which have been used for understanding the determinants of protein stability are small, monomeric, single domain globular proteins like RNase A, Lysozyme and Myoglobin. On the other hand large proteins often show complex unfolding transition profiles that are rarely reversible. The major part of this thesis is focused on studying potential stabilizing/destabilizing interactions in small and large globular proteins. These interactions have been identified and characterized by exploring the effects of various rationally designed mutations on protein stability. Spectroscopic, molecular biological and calorimetric techniques were employed to understand the relationships between protein sequence, structure and stability. The experimental systems used are Leucine binding proteins, Leucine isoleucine valine binding protein (LIVBP), Maltose binding protein (MBP), Ribose binding protein (RBP) and Thioredoxin (Trx). The last section of the thesis discusses thermodynamic properties of molten globule states of the periplasmic protein LBP, LIVBP, MBP and RBP. The amino acid Pro is unique among all the twenty naturally occurring amino acids. In the case of proline, the Cδ of the side chain is covalently linked with the main chain nitrogen atom in a five membered ring. Therefore, Pro lacks amide hydrogen and it is not able to form a main chain hydrogen bond with a carbonyl oxygen. Hence Pro is typically not found in the hydrogen bonded, interior region of α-helix. There have been several studies which showed that introduction of the Pro residue into the interior of an α-helix is destabilizing. Although, it is not common to find Pro residue in the interiors of an α-helix, it has been reported that it occurs with appreciable frequency (14%). The thermodynamic effects of replacements of Pro residue in helix interiors of MBP were investigated in Chapter 2 of this thesis. Unlike many other small proteins, MBP contains 21 Pro residues distributed throughout the structure. It contains three residues in the interiors of α-helices, at positions 48, 133 and 159. These Pro residues were replaced with an alanine and serine amino acids using site directed mutagenesis. Stabilities of all the mutant and wild type proteins have been studied via isothermal chemical denaturation at pH 7.4 and thermal denaturation as a function of pH ranging from pH 6.5 to 10.4. It has been observed that replacement of a proline residue in the middle of an α-helix does not always stabilize a protein. It can be stabilizing if the carbonyl oxygen of residue (i-3) or (i-4) is well positioned to form a hydrogen bond with the ith (mutated) residue and the position of mutation is not buried or conserved in the protein. Partially exposed position have the ability to form main chain hydrogen bonds and Ala seems to be a better choice to substitute Pro than Ser. Unlike other amino acids, the pyrolidine ring of Pro residue imposes rigid constraints on the rotation about the N---Cα bond in the peptide backbone. This causes conformational restriction of the φ dihedral angle of Pro to -63±15º in polypeptides. Therefore, introduction of a rigid Pro residue into an appropriate position in a protein sequence is expected to decrease the conformational entropy of the denatured state and consequently lead to protein stabilization. In Chapter 3 of this thesis, the thermodynamic effects of Pro introduction on protein stability has been investigated in LIVBP, MBP, RBP and Trx. Thirteen single and two double mutants have been generated in the above four proteins. Three of the MBP mutants were characterized by X-ray crystallography to confirm that no structural changes had occurred upon mutation. In the remaining cases, CD spectroscopy was used to show the absence of structural changes. Stability of all the mutant and wild type proteins was studied via isothermal chemical denaturation at neutral pH and thermal denaturation as a function of pH. The mutants did not show enhanced stability with respect to chemical denaturation at room temperature. However, six of the thirteen single mutants showed a small but significant increase in the free energy of thermal unfolding in the range of 0.3-2.4 kcal/mol, two mutants showed no change and five were destabilized. In five of the six cases, the stabilization was because of a reduced entropy of unfolding. Two double mutants were constructed. In both cases, the effects of the single mutations on the free energy of thermal unfolding were non-additive. In addition to the hydrogen bond, hydrophobic and electrostatic interactions, other interactions like cation-π and aromatic-aromatic interactions etc. are also considered to make important contributions to protein stability. The relevance of cation-π interaction in biological systems has been recognized in recent years. It has been reported that positively charged amino acids (Lys, Arg and His) are often located within 6 Å of the ring centroids of aromatic amino acids (Phe, Tyr and Trp). The importance of cation-π interaction in protein stability has been suggested by previous theoretical and experimental studies. We have attempted to determine the magnitude of cation-π interactions of Lys with aromatic amino acids in four different proteins (LIVBP, MBP, RBP and Trx) in Chapter 4 of the thesis. Cation-π pairs have been identified by using the program CaPTURE. We have found thirteen cation-π pairs in five different proteins (PDB ID’s 2liv, 1omp, 1anf, 1urp and 2trx). Five cation-π pairs were selected for the study. In each pair, Lys was replaced with Gln and Met. In a separate series of experiments, the aromatic amino acid in each cation-π pair was replaced by Leu. Stabilities of wild type (WT) and mutant proteins were characterized by similar methods, to those discussed in previous chapters. Gln and Aromatic → Leu mutants were consistently less stable than the corresponding Met mutants reflecting the non-isosteric nature of these substitutions. The strength of the cation-π interaction was assessed by the value of the change in the free energy of unfolding (ΔΔG0=ΔG0 (Met) - ΔG0(WT)). This ranged from +1.1 to –1.9 kcal/mol (average value – 0.4 kcal/mol) at 298 K and +0.7 to –2.6 kcal/mol (average value –1.1 kcal/mol) at the Tm of each WT. It therefore appears that the strength of cation-π interactions increases with temperature. In addition, the experimentally measured values are appreciably smaller in magnitude than the calculated values with an average difference |ΔG0expt -ΔG0calc|avg of 2.9 kcal/mol. At room temperature, the data indicate that cation-π interactions are at best weakly stabilizing and in some cases are clearly destabilizing. However at elevated temperatures, close to typical Tm’s, cation-π interactions are generally stabilizing. In Chapter 5, we have attempted to characterize molten globule states for the periplasmic proteins LBP, LIVBP, MBP and RBP. It was observed that all these proteins form molten globule states at acidic pH (3 - 3.4). All these molten globule states showed cooperative thermal transitions and bound with their ligand comparable to (LBP and LIVBP) or with lower (MBP and RBP) affinity than the corresponding native states. Trp, ANS fluorescence and near-UV CD spectra for ligand bound and free forms of molten globule states were found to be very similar. This shows that molten globule states of these proteins have the ability to bind to their corresponding ligand without conversion to the native state. All four molten globule states showed destabilization relative to the native state. ΔCp values indicate that these molten globule states contain approximately 29-67% of tertiary structure relative to the native state. All four proteins lack prosthetic groups and disulfide bonds. Therefore, it is likely that molten globule states of these proteins are stabilized via hydrophobic and hydrogen bonding interactions.

The structure of the liganded form of phosphate-binding protein, an essential component of the Pst inorganic phosphate active transport system of E. coli has been determined and refined at 1.7 A resolution to an R-factor of 14.7% for 32,222 reflections. The initial model was derived by fitting the amino acid sequence to a 2.5 A resolution electron-density map computed with isomorphous replacement phases from a single iodo derivative with anomalous dispersion data. The mean figure of merit is 0.69 for 11,477 reflections. The model consists of all 321 amino acid residues (2,439 non-hydrogen atoms), the phosphate substrate and 259 ordered water molecules. The molecule is ellipsoid with overall dimensions of 35 A $\times$ 40 A $\times$ 70 A. Phosphate-binding protein structurally resembles six other periplasmic binding proteins (specific for sc L-arabinose, sc D-galactose/ sc D-glucose, leucine/isoleucine/valine, leucine, sulfate and maltose) solved in our laboratory. The molecule consists of two domains, each with a central $\beta$-sheet flanked by $\alpha$-helices. The domains are connected by a hinge which is composed of three strands and one helix. The substrate is bound in the cleft formed between the domains. The general folding pattern is parallel $\alpha$/$\beta$ with the exception of one antiparallel strand in each $\beta$-sheet as a result of the crossover between the domains. The bound phosphate anion is inaccessible to the bulk solvent. It interacts with the protein through twelve hydrogen bonds. Molecular dynamics simulations using free energy perturbation techniques reproduce the experimentally observed specificity of phosphate-binding protein. A decreased affinity is almost entirely due to differences in electrostatic interactions. Electrostatic field calculations based on the finite difference method compute a strong field gradient for the binding site in the absence of the substrate, caused by the proximity of several ionized side chains as well as a number of peptide amides and polar side chains. The gradient is complementary to the net dipole moment of the bound HPO$\sb4\sp{=}$. The electrostatic potential at the binding site is positive averaging about 100 kT/e despite the uncompensated charge of Asp56. (Abstract shortened with permission of author.)

Diaminopropionate ammonia lyase (DAPAL) which belongs to the  class of PLP enzymes is reported only from prokaryotes. It is involved in the removal of two amino groups from its substrate, diaminopropionate, to form ammonia and pyruvate. DAPAL from Escherichia coli (eDAPAL) and Salmonella typhimurium (sDAPAL) was cloned, over expressed and purified using either affinity chromatography or conventional procedures. It was observed that eDAPAL (90 units / mg) was comparatively less active than sDAPAL (200 units / mg). Also the enzymes with the N-terminal His tag were found to be many fold less active than the enzymes without tag. DAPAL had a characteristic absorption maximum at 414nm due to the Schiff`s linkage between PLP and the € - amino group of the active site lysine residue. The apoenzyme was prepared by reaction with L-cysteine, and the resulting thiazolidine complex was easily dialyzed. On reconstitution with PLP, complete regain of absorption spectrum and 60% activity was seen. All the three enzymes (apo-, holo and reconstituted), when subjected to gel filtration chromatography were found to be homodimers of 88 kDa. The active site lysine 78 was mutated to glutamine, and the enzyme was purified to homogeneity. In the mutant enzyme PLP continued to be bound at the active site, but in a different orientation with an absorbance maximum at 406nm. The K78Q enzyme had negligible activity as compared to the wild type enzyme confirming the role of K78 in catalysis. Only a few of the enzymes of the  class have been investigated for their unfolding pathways. Urea induced unfolding studies on sDAPAL revealed that at lower concentrations of urea there was a loss in activity due to the disruption of Schiff's linkage. No gross conformational changes were observed at these concentrations of urea as seen from fluorescence and gel filtration experiments. Increase in concentration of urea led to unfolding of the protein thereby causing a shift in fluorescence maximum from 340nm to 357 nm due to the exposure of the buried tryptophans to the less hydrophobic environment. A considerable amount of aggregation was seen at intermediate urea concentrations, which was possibly the reason for the inability of the protein to refold completely. Based on the results, a concerted mechanism for dissociation and unfolding was proposed for sDAPAL. Aminooxy compounds, which are mechanism-based inhibitors for PLP enzymes have been used as drugs against various disorders for the last few decades. In order to probe the mechanism and efficiency with which these compounds inhibit sDAPAL, cycloserine (D and L), methoxyamine (MA) and aminooxyacetic acid (AAA) were chosen for the inhibition studies. The inhibition rates were measured by monitoring decrease in absorbance at 414nm, increase in the range of 320-330nm due to the product formation and loss of activity upon incubation with the inhibitor. It was seen that both the enantiomers of cycloserine were equally effective in disrupting the Schiff’s linkage with the second order rate constants of 15.8 and 36 M -1 sec –1 respectively. Spectral measurements showed two isosbestic points in the case of DCS and one in the case of LCS. Product of this inhibition reaction was identified to be a heat and acid stable compound namely a hydroxyisooxazole derivative of PMP. It was similar in nature to that reported from GABA aminotransferase. These results showed that unlike in the case of alanine racemase, sDAPAL could be inhibited equally well by both the enantiomers. The inhibition studies with the other two inhibitors namely AAA and MA, showed AAA to be more efficient at disrupting the Schiff’s linkage and causing inactivation of the enzyme. The visible absorbance spectrum showed a single isosbestic point in both the cases, indicative of a single step involved in the formation of the final product. The elution profile of the product of the enzymatic as well as non-enzymatic reactions on a C-18 HPLC column was similar and the product was identified to be an oxime. These inhibitors reacted with sDAPAL many fold better than the other PLP dependent enzymes and therefore these compounds can serve as potential drugs for sDAPAL.

Occasionally, crystallisation of proteins works in mysterious ways! One might obtain crystals of a protein of unknown identity in place of the protein for which crystallisation experiments were performed. If the investigator is not aware of such possibilities, valuable time and resources might be lost in attempting to determine the structure of such proteins. Instances of non-target protein getting crystallised may not come to light at all or may be realised only when attempts to determine the structure completely fail by conventional procedures after collecting and processing the diffraction data. Usually, it is not possible to reproduce the crystals of the same protein as their occurrence is serendipitous. Such rare instances of crystallisation are probably caused by fluctuating environmental or crystallisation conditions and are not reproducible. It could also be due to contaminating microbes, which is more likely when the experimentalist is not well experienced. Therefore, experimental phasing of the data collected on serendipitously obtained crystals could be a challenging task. With the rapid increase in the number of structures deposited in the protein data bank (PDB), molecular replacement has become the method of choice for structure determination in macromolecular X-ray crystallography. This is due to the fact that it is possible to select a suitable phasing model for most target proteins based on their sequence information. However, if the identity of the target protein itself is uncertain, all attempts of structure determination using phasing models selected on the basis of target protein sequence-dependent search would fail. Sequence-independent ab initio phasing techniques such as ARCIMBOLDO (Meindl et al., 2012), which has recently become available, could provide leads only if the non-target protein is an all-α-protein and the associated diffraction data extends to a resolution better than 2 Å. Even then, the success rate with this technique is low. Hence, it becomes important to employ a sequence-independent method of structure determination for such mysteriously obtained crystals. This thesis reports crystal structures of proteins which are serendipitously crystallised using a large-scale application of Molecular Replacement (MR) technique (referred in this thesis as MarathonMR). This thesis also presents an evaluation of molecular replacement strategies for structure determination. The thesis begins with an overview of crystallographic methods of structure determination with an emphasis on the method of molecular replacement (Chapter 1). The most prominent of the results obtained in the course of these investigations pertains to a crystal obtained during routine crystallisation of a viral protein mutant in the year 2011. The cell parameters were different from cell constants of crystals obtained with other known viral protein mutants crystallised earlier in the same laboratory. Unfortunately, this crystal could not be reproduced in the same form in subsequent crystallisation trials. All attempts to determine the structure through conventional molecular replacement techniques using a combination of domains from a nearly identical virus coat protein protomer as the phasing model had failed. The data was shelved as “not-solvable” in late 2011. However, the crystal had diffracted to 1.9 Å and had excellent merging statistics. Therefore, the data was retrieved recently and additional attempts were made to determine the structure through phasing techniques that have become available recently. Techniques such as AMPLE (Bibby et al., 2013) and Rosetta (DiMaio, 2013), which use large-scale homology models coupled with molecular replacement, did not lead to meaningful solutions. A couple of helices identified by ARCIMBOLDO (Meindl et al., 2012) were neither correct (retrospectively) nor sufficient to determine the entire structure. Given the excellent merging statistics of the crystal data, there was significant motivation to determine the structure, though it meant developing a fresh protocol. It was at this time that we came across the work of Stokes-Rees and Sliz (2010) in which they had demonstrated that it is possible to determine structure of proteins of unknown identity by employing almost every known protein structure as a potential phasing model. The work reported in the thesis is a result of an earlier project to examine the relationship between properties of phasing models and the quality of target protein model generated through MR by employing large scale molecular replacement runs. This project was initiated because of the realisation that the recent explosion in crystallographic structural studies has resulted in near complete exploration of the “fold-space” of proteins and PDB now has a representative structure for most plausible folds of proteins. Some folds are highly represented in the PDB. Hence, it is likely that there would be at least one homologue in the PDB which could be used as a phasing model to successfully determine the structure of a protein of unknown identity if the diffraction dataset is of excellent quality. Hence, the single dataset which had diffracted to 1.9 Å resolution was used to develop a MarathonMR procedure for structure determination. MarathonMR procedure takes sequence-independent approach to structure determination and employs large-scale molecular replacement calculations to identify the closest homologue (in structural terms initially). This protocol is described in Chapter 2 (Materials and methods) of the thesis. Through MarathonMR, structure of the dataset which had remained unsolved for 5 years was finally determined. Nearly complete sequence of the polypeptide could be deduced by inspecting the electron density map due to the high resolution and quality of the map. The protein was found to be a phosphate binding protein from a soil bacterium Stenotrophomonas maltophilia (SmPBP). The way in which the structure was determined and possible explanations for the mysterious source of this protein which had crystallised instead of the target protein is discussed in Chapter 3. Though MarathonMR procedure was developed to solve a single dataset, it was soon realised that the same procedure could be applied to other similar datasets, all of which had diffracted to reasonable resolutions with good merging statistics but had remained unsolved for unknown reasons. Among such datasets, one of the datasets which was collected in 2007 and had diffracted to 2.3 Å resolution had cell parameters very close to that of SmPBP. Hence, a poly-alanine model of the structure of SmPBP, which was determined by then, was used as the phasing model to run molecular replacement and the structure was readily solved. It was surprising to note that SmPBP had crystallised serendipitously not once but twice, once in 2011 resulting in crystals that diffracted to 1.9 Å resolution and earlier in 2007 in crystals that diffracted to 2.3 Å resolution independently by two different investigators in the same laboratory. Both the structures are nearly identical and a comparison of these structures is presented in Chapter 4. Structure of SmPBP determined at 2.3 Å resolution by MarathonMR also corresponds to the dataset that had remained unsolved for the longest period of time (9 years). This success of structure determination after the lapse of such a long period emphasises the importance of carefully preserving X-ray diffraction data irrespective of its immediate outcome. In Chapter 5 of the thesis, another instance of non-target protein crystallisation, the structure of which was determined using the MarathonMR procedure is described. The crystal was obtained while carrying out crystallisation of mutants of a survival protein (SurE) expressed in Salmonella typhimurium when the bacterium is subjected to environmental or internal stresses. The original investigator had used the structure of SurE as the phasing model to determine structure of the mutant crystals and obtained a model with R and Rfree of 35% and 40%, respectively. However, the model did not refine further to lower R-factors suggesting that the solution obtained may not be correct. MarathonMR indicated that the fold of the crystallised protein could be similar to that of glycerol dehydrogenase. As SurE shares some fold similarity with one of the domains of GlyDH, the original investigator might have been able to achieve a limited success with R/Rfree factors of 35% and 40%, respectively. As the merging statistics for this diffraction data set was poor, the diffraction images were reprocessed in XDS program on Xia2 automated spot processing pipeline. The data statistics indicated merohedral twinning (14%). However, using appropriate parameters, it was possible to refine the structure obtained by MarathonMR to acceptable R/Rfree using the Refmac program. Four protomers were present in the crystal asymmetric unit (ASU). Non-crytsallographic symmetry averaging of electron density over these four molecules further improved the electron density. As the data was limited to 2.7 Å resolution, it was not possible to deduce the identity of every residue of the protein unambiguously based solely on the resulting electron density map. With the identity of the amino acids that could be deduced with certainty, it was clear that the protein belongs to glycerol dehydrogenase from a species of Enterobacteriacea family. Though a similar structure of glycerol dehydrogenase has been reported from Serratia, there are clear differences in many unambiguously determined residues which suggest that the protein is not from Serriatia. The protein has been named EnteroGlyDH as the source of the protein is likely to be from a species of Enterobacteriacea family. The structure of the protein, its biochemical implications and possible reasons for the serendipitous crystallisation of a non-target are discussed. Chapter 6 discusses the structure determination of an inorganic pyrophosphatase and catalytic domain of Succinyl transferase, the crystals of which had diffracted to 2.3 Å and 3.1 Å, respectively, but had remained unsolved. Neither of the datasets corresponds to the intended target proteins. The dataset corresponding to the protein whose structure was determined as that of an inorganic pyrophosphatase was provided by a colleague from a different laboratory in the Indian Institute of Science. It is interesting to note that the investigator had carried this dataset to one of the CCP4 workshops and had tried to determine the structure with the help of experts in the workshop. The attempts to determine its structure had however failed for reasons that are obvious now. The original investigator was unfortunately making efforts with an erroneous assumption on the identity of the target protein. As these enzymes are well studied, their structures and functions are briefly discussed. It is already well established that molecular replacement is being used with increasing frequency as the phasing technique when compared to other experimental phasing techniques. With the ever growing number of structures in the PDB, high population of certain folds and a near-plateau attained in the identification and growth of new folds, it is reasonable to expect that molecular replacement will be used even more frequently in the years to come. Therefore, for carrying out molecular replacement for a given diffraction dataset of a target protein, it is very likely that several homologous structures would be available in the PDB that could be used as potential phasing models. Hence, it becomes important to understand the influence of phasing model on the quality and accuracy of model generated through MR to achieve the best structure solution. To understand this relationship between phasing model and model obtained by MR protocol, re-determination of already known structures deposited in the PDB starting with their respective structure factors and various phasing models was initiated. Structures belonging to TIM beta/alpha-barrel (SCOPe ID: c.1) and Lysozyme-like (SCOPe ID: d.2) folds were chosen as targets. The structure of each target was re-determined serially starting with poly-alanine models of all available unique homologues as phasing models. Due to the multi-dimensional nature of this study, the results obtained were represented in a graphical form with nodes and edges. Detailed methodology of the work carried out and the data representation model are discussed in the Chapter 2 (Materials and methods). It was found that after a certain sequence identity cut-off, sequence identity between phasing model and target seems to have little influence on the quality and accuracy of the model generated through MR. Instead, other qualities of the phasing model such as Rfree and RSCC influence the quality of MR models. These results are discussed in Chapter 7. Learning from the work reported in this thesis are discussed in concluding chapter. The possible logical and programmatic upgrades to MarathonMR protocol and future path in which the relationship between phasing models and models generated through MR can be studied are discussed in Chapter 8 (Conclusion and future prospects).