Mycobacterial Stress Regulation: The Dps ``Twin Sister'' Defense Mechanism and Structure-Function Relationship

In this work, we have tried to emphasize the connection between mycobacterial growth and regulation of gene expression. Utilization of multiple carbon sources and diauxic growth helps bacteria to regulate gene expression at an optimum level so that the inhospitable conditions encountered during nutrient depletion can be circumvented. These aspects will be discussed with respect to mycobacterial growth in subsequent sections. Identification and characterization of genes induced under such conditions is helpful to understand the physiology of the bacterium. Although it is necessary to compare the total expression profile of proteins as they transit from vegetative growth to stationary phase, at times a lot of insights can be deciphered from the expression pattern of one or two proteins. We have compared the protein expression and sigma factor selectivity of two such proteins in M. smegmatis to understand the differential regulation of genes playing diverse function in the same species. Some newer insights on the structure and function of one of the Dps proteins are also explained.

Allostery and conformational free energy changes in human tryptophanyl-tRNA synthetase from essential dynamics and structure networks

The interdependence of the concept of allostery and enzymatic catalysis, and they being guided by conformational mobility is gaining increased prominence. However, to gain a molecular level understanding of llostery and hence of enzymatic catalysis, it is of utter importance that the networks of amino acids participating in allostery be deciphered. Our lab has been exploring the methods of network analysis combined with molecular dynamics simulations to understand allostery at molecular level. Earlier we had outlined methods to obtain communication paths and then to map the rigid/flexible regions of proteins through network parameters like the shortest correlated paths, cliques, and communities. In this article, we advance the methodology to estimate the conformational populations in terms of cliques/communities formed by interactions including the side-chains and then to compute the ligand-induced population shift. Finally, we obtain the free-energy landscape of the protein in equilibrium, characterizing the free-energy minima accessed by the protein complexes. We have chosen human tryptophanyl-tRNA synthetase (hTrpRS), a protein esponsible for charging tryptophan to its cognate tRNA during protein biosynthesis for this investigation. This is a multidomain protein exhibiting excellent allosteric communication. Our approach has provided valuable structural as well as functional insights into the protein. The methodology adopted here is highly generalized to illuminate the linkage between protein structure networks and conformational mobility involved in the allosteric mechanism in any protein with known structure.

The influence of impurities on calcium phosphate floc structure and size in sugar solutions

Settling, dewatering and filtration of flocs are important steps in industry to remove solids and improve subsequent processing. The influence of non-sucrose impurities (Ca2+, Mg2+, phosphate and aconitic acid) on calcium phosphate floc structure (scattering exponent, Sf), size and shape were examined in synthetic and authentic sugar juices using X-ray diffraction techniques. In synthetic juices, Sf decreases with increasing phosphate concentration to values where loosely bound and branched flocs are formed for effective trapping and removal of impurities. Although, Sf did not change with increasing aconitic acid concentration, the floc size significantly decreased reducing the ability of the flocs to remove impurities. In authentic juices, the flocs structures were marginally affected by increasing proportions of non-sucrose impurities. However, optical microscopy indicated the formation of well-formed macro-floc network structures in sugar cane juices containing lower proportions of non-sucrose impurities. These structures are better placed to remove suspended colloidal solids.

Vertex-Fused Metallaborane Clusters: Synthesis, Characterization and Electronic Structure of [(eta(5)-C5Me5Mo)(3)MoB9H18]

The reaction of the [(eta(5)-C5Me5)MoCl4] complex with [LiBH4 - TH F] in toluene at - 70 degrees C, followed by pyrolysis at 110 degrees C, afforded dark brown [(eta(5)-C5Me5Mo)(3)MoB9H18], 2, in parallel with the known [(eta(5)-C5Me5Mo)(2)B5H9], 1. Compound 2 has been characterized in solution by H-1, B-11, and C-13 NMR spectroscopy and elemental analysis, and the structural types were unequivocally established by crystallographic studies. The title compound represents a novel class of vertex-fused clusters in which a Mo atom has been fused in a perpendicular fashion between two molybdaborane clusters. Electronic structure calculations employing density functional theory yield geometries in agreement with the structure determinations, and on grounds of density functional theory calculations, we have analyzed the bonding patterns in the structure,

Understanding solvent effects on structure and reactivity of organic intermediates: a Raman study

The effect of solvent on chemical reactivity has generally been explained on the basis of the dielectric constant and viscosity. However a number of spectroscopic studies, including UV-VIS, IR and Raman, has led to numerous empirical parameters to define solvent effect based on either solvating ability or polarity scale. These parameters include solvent polarizability, dipolarity, Lewis acidity and Lewis basicity, E-T(30), pi*, alpha, beta etc. However, from a structural point of view, we can separate solvation as static and dynamic processes. The static solvation basically relates to stabilization of the molecular structure by the solvent to attain the equilibrium structure, both in the intermediate and ground state. Dynamic solvation relates to solvent reorganization-induced dynamics prior to the structural reorganization to reach the equilibrium state. In this paper, we present (a) structural distortions induced by the solvent due to preferential solvation of the triplet excited state, and (b) the importance of dynamic solvation induced by vibronic coupling (pseudo-Jahn-Teller coupling). The examples include the effect of solvent on structure and reactivity of excited states of 2,2,2-trifluoroacetophenone (TFA). Based on the comparison of time resolved resonance Raman (TR3) data of TFA and other substituted acetophenone systems, it was found that change in solvent polarity indeed results in electronic state switching and structural changes in the excited state, which explains the trend in reactivity. Further, a TR3 study of fluoranil (FA) in the triplet excited state in solvents of varying polarities indicates that the structure of FA in the triplet excited state is determined by vibronic coupling effects and thus distorted structure. These experimental results have been well supported by density functional theoretical computational studies.

Structure of Mycobacterium tuberculosis RuvA, a protein involved in recombination

The process of recombinational repair is crucial for maintaining genomic integrity and generating biological diversity. In association with RuvB and RuvC, RuvA plays a central role in processing and resolving Holliday junctions, which are a critical intermediate in homologous recombination. Here, the cloning, purification and structure determination of the RuvA protein from Mycobacterium tuberculosis (MtRuvA) are reported. Analysis of the structure and comparison with other known RuvA proteins reveal an octameric state with conserved subunit-subunit interaction surfaces, indicating the requirement of octamer formation for biological activity. A detailed analysis of plasticity in the RuvA molecules has led to insights into the invariant and variable regions, thus providing a framework for understanding regional flexibility in various aspects of RuvA function.

Formation of a novel surface structure encoded by Salmonella Pathogenicity Island 2.

The type III secretion system (T3SS) encoded by Salmonella Pathogenicity Island 2 (SPI2) is essential for virulence and intracellular proliferation of Salmonella enterica. We have previously identified SPI2-encoded proteins that are secreted and function as a translocon for the injection of effector proteins. Here, we describe the formation of a novel SPI2-dependent appendage structure in vitro as well as on the surface of bacteria that reside inside a vacuole of infected host cells. In contrast to the T3SS of other pathogens, the translocon encoded by SPI2 is only present singly or in few copies at one pole of the bacterial cell. Under in vitro conditions, appendages are composed of a filamentous needle-like structure with a diameter of 10 nm that was sheathed with secreted protein. The formation of the appendage in vitro is dependent on acidic media conditions. We analyzed SPI2-encoded appendages in infected cells and observed that acidic vacuolar pH was not required for induction of SPI2 gene expression, but was essential for the assembly of these structures and their function as translocon for delivery of effector proteins.

The 310 helical conformation of a pentapeptide containing a-aminoisobutyric acid (Aib): X-ray crystal structure of Tos-(Aib)5 OMe

The pentapeptide Tos-(Aib)5-OMe adopts a 310 helical conformation in the solid state, with three consecutive Type III B-turns stabilized by intramolecular hydrogen bonds.

The crystal and molecular structure of benzyloxycarbonyl-a-amino-isobutyryl-L-prolyl-methylamide. The observation of an X2-Pro3 Type III b-turn

The crystal and molecular structure of N-benzyloxycarbonyl-a-aminoisobutyryl-L-prolyl methylamide, the amino terminal dipeptide fragment of alamethicin, has been determined using direct methods. The compound crystallizes in the orthorhombic system with the space group P212-21. Cell dimensions are a = 7.705 A, b = 11.365 A, and c = 21.904 A. The structure has been refined using conventional procedures to a final R factor of 0.054. The molecular structure possesses a 4 - 1 intramolecular N-H--0 hydrogen bond formed between the CO group of the urethane moiety and the NH group of the methylamide function. The peptide backbone adopts the type 111 P-turn conformation, with 42 = -51.0°, +* = -39.7",&j = -65.0', $3 = -25.4'. An unusual feature is the occurrence of the proline residue at position 3 of the P-turn. The observed structure supports the view that Aib residues initiate the formation of type 111 @-turn conformations. The pyrrolidine ring is puckered in Cy-exo fashion.

X-Pro peptides: Synthesis and solution conformation of benzyloxycarbonyl-(Aib-Pro)4methyl ester. Evidence for a novel helical structure

The synthesis of the octapeptide, benzyloxycarbonyl-(-aminoisobutyryl-L-prolyl)4-methyl ester [Z-(Aib-Pro)4-OMe] and an analysis of its solution conformation is reported. The octapeptide is shown to possess three strong intramolecular hydrogen bonds on the basis of studies of the solvent and temperature dependence of NH chemical shifts and rates of hydrogen-deuterium exchange. 13C studies are consistent with a structure involving only trans Aib-Pro bonds, while ir experiments support a hydrogen-bonded conformation. The Aib 3, 5, and 7 NH groups are shown to participate in hydrogen bonding. A 310 helical conformation compatible with the spectroscopic data is suggested. The proposed conformation consists of three type III -turns with Aib and Pro at the corners and stabilized by 4 1 intramolecular hydrogen bonds.

Use of structure-directed DNA ligands to probe the binding of recA protein to narrow and wide grooves of DNA and on its ability to promote homologous pairing

We have used circular dichroism and structure-directed drugs to identify the role of structural features, wide and narrow grooves in particular, required for the cooperative polymerization, recognition of homologous sequences, and the formation of joint molecules promoted by recA protein. The path of cooperative polymerization of recA protein was deduced by its ability to cause quantitative displacement of distamycin from the narrow groove of duplex DNA. By contrast, methyl green bound to the wide groove was retained by the nucleoprotein filaments comprised of recA protein-DNA. Further, the mode of binding of these ligands and recA protein to DNA was confirmed by DNaseI digestion. More importantly, the formation of joint molecules was prevented by distamycin in the narrow groove while methyl green in the wide groove had no adverse effect. Intriguingly, distamycin interfered with the production of coaggregates between nucleoprotein filaments of recA protein-M13 ssDNA and naked linear M13 duplex DNA, but not with linear phi X174 duplex DNA. Thus, these data, in conjunction with molecular modeling, suggest that the narrow grooves of duplex DNA provide the fundamental framework required for the cooperative polymerization of recA protein and alignment of homologous sequences. These findings and their significance are discussed in relation to models of homologous pairing between two intertwined DNA molecules.

The Use of Mesenchymal Stromal Cells in the Treatment of Diseases of the Cornea

As a key component of the ocular surface required for vision, the cornea has been extensively studied as a site for cell and tissue-based therapies. Historically, these treatments have consisted of donor corneal tissue transplants, but cultivated epithelial autografts have become established over the last 15 years as a routine treatment for ocular surface disease. Ultimately, these treatments are performed with the intention of restoring corneal transparency and a smooth ocular surface. The degree of success, however, is often dependent upon the inherent level of corneal inflammation at time of treatment. In this regard, the anti-inflammatory and immuno-modulatory properties of mesenchymal stromal cells (MSC) have drawn attention to these cells as potential therapeutic agents for corneal repair. The origins for MSC-based therapies are founded in part on observations of the recruitment of endogenous bone marrow-derived cells to injured corneas, however, an increasing quantity of data is emerging for MSC administered following their isolation and ex vivo expansion from a variety of tissues including bone marrow, adipose tissue, umbilical cord and dental pulp. In brief, evidence has emerged of cultured MSC, or their secreted products, having a positive impact on corneal wound healing and retention of corneal allografts in animal models. Optimal dosage, route of administration and timing of treatment, however, all remain active areas of investigation. Intriguingly, amidst these studies, have emerged reports of MSC transdifferentiation into corneal cells. Clearest evidence has been obtained with respect to expression of markers associated with the phenotype of corneal stromal cells. In contrast, the evidence for MSC conversion to corneal epithelial cell types remains inconclusive. In any case, the conversion of MSC into corneal cells seems unlikely to be an essential requirement for their clinical use. This field of research has recently become more complicated by reports of MSC-like properties for cultures established from the peripheral corneal stroma (limbal stroma). The relationship and relative value of corneal-MSC compared to traditional sources of MSC such as bone marrow are at present unclear. This chapter is divided into four main parts. After providing a concise overview of corneal structure and function, we will highlight the types of corneal diseases that are likely to benefit from the anti-inflammatory and immuno-modulatory properties of MSC. We will subsequently summarize the evidence supporting the case for MSC-based therapies in the treatment of corneal diseases. In the third section we will review the literature concerning the keratogenic potential of MSC. Finally, we will review the more recent literature indicating the presence of MSC-like cells derived from corneal tissue.