Dissertations / Theses on the topic 'Periplasmic binding protein (PBP)'

La bactérie Agrobacterium tumefaciens établit une relation à long terme avec les plantes. Elle peut avoir deux modes de vie : (1) pathogène lorsqu’elle possède le plasmide de virulence Ti (Tumor inducing) provoquant la maladie de la galle du collet caractérisée par la formation de tumeur chez la plante, (2) non pathogène vivant dans la rhizosphère. Dans ces deux modes de vie, Agrobacterium utilise les PBP (protéines périplasmiques de liaision) associées à des transporteurs ABC pour importer des molécules issues de plante servant de nutriments. La PBP sélectionne et fixe le ligand qu’elle apporte au transporteur ABC, qui permet son passage dans le cytoplasme grâce à l’hydrolyse de deux molécules d’ATP. La spécificité du transporteur entier dépend de la PBP. Les molécules transportées et dégradées par la bactérie apparaissent comme un avantage trophique pour la colonisation de l’environnement.Lorsqu’A. tumefaciens est pathogène, la bactérie transfère une partie de son plasmide Ti dans le génome des cellules végétales, induisant la production et sécrétion par la plante de composés spécifiques pour la bactérie, appelés opines. Une vingtaine d’opines sont connues à ce jour, et chacune d’elles peut être métabolisée par des souches d’A. tumefaciens. La souche B6 possède un pTi de type octopine, qui porte les gènes de métabolisme de la famille des mannityl-opines, composée de l’acide agropinique, l’agropine, l’acide mannopinique et la mannopine. D’après des études génétiques chez des souches de type octopine, les systèmes PBP-transporteur ABC associés sont respectivement AgaABCD, AgtABCD, MoaABCD et MotABCD.Dans la rhizosphère, les graines en germination influencent la composition de la rhizosphère et favorisent la croissance de microorganismes en libérant des molécules telles que les sucres de la famille du raffinose (RFO, Raffinose Family of Oligosaccharides). Des analyses in silico d’Agrobacterium fabrum C58 indiquent que la souche possèderait un opéron que nous avons appelé mel, proche de l’opéron agp chez Ensifer meliloti 1021 décrit comme responsable du transport et de la dégradation des RFO qui semble influer sur la survie de la bactérie dans la rhizosphère. Nous avons fait l’hypothèse que la PBP MelB de l’opéron mel chez A. fabrum C58 est responsable du transport des RFO.Mon travail de thèse a permis de finaliser la caractérisation structurale et biochimique des transporteurs des mannityl-opines et a permis l’identification et la caractérisation de MelB comme responsable de l’import des RFO et du galactinol (précurseur des RFO). Le transport et la dégradation des molécules transportées par ces PBP sont importants pour la colonisation de leur environnement
Agrobacterium tumefaciens bacterium establishes a long-term relationship with plants. It can have two lifestyles: (1) pathogenic when it harbors the virulence plasmid Ti (Tumor inducing) causing the crown gall disease characterized by tumor formation in plant, (2) non-pathogenic living in the rhizosphere. In these two lifestyles, Agrobacterium uses PBP (periplasmic binding proteins) associated with ABC transporters to import molecules from plants as nutrients. PBP selects and binds the ligand that it brings to the ABC transporter, which allows its passage into the cytoplasm by the hydrolysis of two ATP molecules. The specificity of the entire transporter depends on PBP. The molecules transported and degraded by the bacteria appear as a trophic advantage for the colonization of the environment.When A. tumefaciens is pathogenic, the bacterium transfers part of its Ti plasmid into the genome of plant cells, inducing the production and secretion by the plant of specific compounds for the bacterium, called opines. Twenty opines are known to date, and each of them can be metabolized by A. tumefaciens strains. The strain B6 possesses an octopine-type pTi, which harbors metabolism genes of the mannityl-opines family, composed of agropinic acid, agropine, mannopinic acid and mannopine. According to genetic studies in octopine type strains, the associated PBP-transporter ABC systems are AgaABCD, AgtABCD, MoaABCD and MotABCD, respectively.In the rhizosphere, germinating seeds influence the composition of the rhizosphere and promote the growth of microorganisms by releasing molecules such as Raffinose Family of Oligosaccharides (RFO). In silico analyzes of Agrobacterium fabrum C58 indicate that the strain possesses an operon we called mel, similar to the agp operon in Ensifer meliloti 1021 described as responsible for the transport and degradation of RFOs that appears to influence the survival of the bacterium in the rhizosphere. We hypothesized that the PBP MelB of the mel operon in A. fabrum C58 is responsible for the transport of the RFOs.My thesis work allowed to finalize the structural and biochemical characterization of mannityl-opines transporters and allowed the identification and characterization of MelB as responsible for the import of RFO and galactinol (precursor of the RFO). The transport and degradation of the molecules transported by these PBPs are important for the colonization of their environment

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.

Penicillin-binding proteins (PBPs) are required in the synthesis of the cell wall of bacteria. In Bacillus subtilis, PBPs play important roles in the life cycle, including both vegetative growth and sporulation, and contribute to the formation of the different structures of vegetative cell wall and spore cortex. The B. subtilis genome sequencing project revealed there were two uncharacterized genes, ykuA and yrrR, with extensive sequence similarity to class B PBPs. These two genes are renamed and referred to henceforth as pbpH and pbpI, respectively.

A sequence alignment of the predicted product of pbpH against the microbial protein database demonstrated that the most similar protein in B. subtilis is PBP2A and in E. coli is PBP2. This suggested that PbpH belongs to a group of the genes required for maintaining the rod shape of the cell. Study of a pbpH-lacZ fusion showed that pbpH was expressed weakly during vegetative growth and the expression reached the highest level at the transition from exponential phase to stationary phase. The combination of a pbpA deletion and the pbpH deletion was lethal and double mutant strains lacking pbpH and pbpC or pbpI (also named yrrR) were viable. The viable mutants were indistinguishable from the wild-type except that the vegetative PG of the pbpC pbpH strain had a slightly slightly lower amount of disaccharide tetrapeptide with 1 amidation and higher amount of disaccharide tripeptide tetrapeptide with 2 amidations when compared to others strains. This suggests that PbpC (PBP3) is involved in vegetative PG synthesis but only affects the PG structure with a very low efficiency.

A pbpA pbpH double mutant containing a xylose-regulated pbpH gene inserted into the chromosome at the amyE locus was constructed. Depletion of PbpH resulted in an arrest in cell growth and a dramatic morphological change in both vegetative cells and outgrowing spores. Vegetative cells lacking pbpA and pbpH expression swelled and cell elongation was arrested, leading to the formation of pleiomorphic spherical cells and eventual lysis. In these cells, cell septations were randomly localized, cell walls and septa were thicker than those seen in wild type cells, and the average cell width and volume were larger than those of cells expressing pbpA or pbpH. The vegetative PG had an increased abundance of one unidentified muropeptide. Spores produced by the pbpA pbpH double mutant were able to initiate germination but the transition of the oval-shaped spores to rod-shape cells was blocked. The outgrowing cells were spherical, gradually enlarged, and eventually lysed. Outgrowth of these spores in the presence of xylose led to the formation of helical cells. Thus, PbpH is apparently required for maintenance of cell shape, specifically for cell elongation. PbpH and PBP2a play a redundant role homologous to that of PBP2 in E. coli.

A sequence alignment of the predicted product of pbpI against the microbial protein database demonstrated that the most similar protein in B. subtilis is SpoVD and in E. coli is PBP3. This suggested that PbpI belongs to the group of the genes required for synthesis of the spore or septum PG. PbpI was identified using radio-labeled penicillin and found to run underneath PBP4 on SDS-PAGE. PbpI is therefore renamed PBP4b. Study of a pbpI-lacZ fusion showed that pbpI was expressed predominantly during early sporulation. A putative sigma F recognition site is present in the region upstream of pbpI and studies using mutant strains lacking sporulation-specific sigma factors demonstrated that the expression of pbpI is mainly dependent on sigma factor F. A pbpI single mutant, a pbpI pbpG double mutant, and a pbpI pbpF double mutant were indistinguishable from the wild-type. The sporulation defect of a pbpI pbpF pbpG triple mutant was indistinguishable from that of a pbpF pbpG double mutant. Structure parameters of the forespore PG in a pbpI spoVD strain are similar to that of a spoVD strain. These results indicate that PBP4b plays a unknown redundant role.

Master of Science

Penicillin-binding proteins (PBP's) are membrane-associated enzymes involved in the polymerization of peptidoglycan. PBP's are divided into three classes based upon their molecular weights and functional domains. Gene expression is regulated in the two differentiated cells in Bacillus subtilis, the mother cell and the forespore, by coordinated expression of different sigma factors that recognize specific promoters in each compartment. The functional and compartmental specificity of individual penicillin-binding proteins from the different classes of PBP's were examined during sporulation in B. subtilis. Analyses of three class A high molecular weight PBP's indicated that pbpF and pbpG must be expressed in the forespore to carry out their specific role during spore peptidoglycan synthesis. Expressing pbpD in either the forespore or the mother cell could not complement for the loss of pbpF and pbpG, suggesting that there must be additional sequence information in PBP2c and PBP2d that allows them to carry out their specific role during germ cell wall synthesis. Analyses of a low molecular weight PBP, PBP5*, suggested that expressing dacB in either the mother cell or in the forespore could regulate the level of spore peptidoglycan cross-linking to what is typical of wild type spore peptidoglycan.
Master of Science

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.

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

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

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.