Effect of signal peptide on stability and folding of escherichia coli thioredoxin

The signal peptide plays a key role in targeting and membrane insertion of secretory and membrane proteins in both prokaryotes and eukaryotes. In E. coli, recombinant proteins can be targeted to the periplasmic space by fusing naturally occurring signal sequences to their N-terminus. The model protein thioredoxin was fused at its N-terminus with malE and pelB signal sequences. While WT and the pelB fusion are soluble when expressed, the malE fusion was targeted to inclusion bodies and was refolded in vitro to yield a monomeric product with identical secondary structure to WT thioredoxin. The purified recombinant proteins were studied with respect to their thermodynamic stability, aggregation propensity and activity, and compared with wild type thioredoxin, without a signal sequence. The presence of signal sequences leads to thermodynamic destabilization, reduces the activity and increases the aggregation propensity, with malE having much larger effects than pelB. These studies show that besides acting as address labels, signal sequences can modulate protein stability and aggregation in a sequence dependent manner.

Affinity of a galactose-specific legume lectin from Dolichos lablab to adenine revealed by X-ray cystallography

Crystal structure analysis of a galactose-specific lectin from a leguminous food crop Dolichos lablab (Indian lablab beans) has been carried out to obtain insights into its quaternary association and lectin-carbohydrate interactions. The analysis led to the identification of adenine binding sites at the dimeric interfaces of the heterotetrameric lectin. Structural details of similar adenine binding were reported in only one legume lectin, Dolichos biflorus, before this study. Here, we present the structure of the galactose-binding D. lablab lectin at different pH values in the native form and in complex with galactose and adenine. This first structure report on this lectin also provides a high resolution atomic view of legume lectin-adenine interactions. The tetramer has two canonical and two DB58-like interfaces. The binding of adenine, a non-carbohydrate ligand, is found to occur at four hydrophobic sites at the core of the tetramer at the DB58-like dimeric interfaces and does not interfere with the carbohydrate-binding site. To support the crystallographic observations, the adenine binding was further quantified by carrying out isothermal calorimetric titration. By this method, we not only estimated the affinity of the lectin to adenine but also showed that adenine binds with negative cooperativity in solution.

Corporate social responsibilities of Indian public sector enterprises-a case study of Bharat Heavy Electricals Limited (BHEL)

The high level of public accountability attached to Public Sector Enterprises as a result of public ownership makes them socially responsible. The Committee of Public Undertakings in 1992 examined the issue relating to social obligations of Central Public Sector Enterprises and observed that ``being part of the `State', every Public Sector enterprise has a moral responsibility to play an active role in discharging the social obligations endowed on a welfare state, subject to the financial health of the enterprise''. It issued the Corporate Social Responsibility Guidelines in 2010 where all Central Public Enterprises, through a Board Resolution, are mandated to create a CSR budget as a specified percentage of net profit of the previous year. This paper examines the CSR activities of the biggest engineering public sector organization in India, Bharath Heavy Electricals Limited. The objectives are twofold, one, to develop a case study of the organization about the funds allocated and utilized for various CSR activities, and two, to examine its status with regard to other organizations, the 2010 guidelines, and the local socio-economic development. Secondary data analysis results show three interesting trends. One, it reveals increasing organizational social orientation with the formal guidelines in place. Two, Firms can no longer continue to exploit environmental resources and escape from their responsibilities by acting separate entities regardless of the interest of the society and Three the thrust of CSR in public sector is on inclusive growth, sustainable development and capacity building with due attention to the socio-economic needs of the neglected and marginalized sections of the society.

NON-GLYCINE RESIDUES IN THE DISALLOWED REGIONS OF THE RAMACHANDRAN MAP AND THEIR NEIGHBORHOOD PREFERENCES

The allowed and the ``disallowed'' regions in the celebrated Ramachandran map (phi-psi] map) was elegantly deduced by Ramachandran, Ramakrishnan and Sasisekharan even before the protein crystal structures became available. This powerful map was derived based on rigid geometry of the peptide group and later several investigations on protein crystal structures reported the occurrence of a small fraction of the phi-psi] torsion angles in the disallowed region. The question is what factors make these residues adopt disallowed conformations? Is it driven by the necessity to maintain the overall topology or is it associated with function or is it just that the disallowed conformations are extreme limits of the allowed conformations? Today, with the availability of a large number of high resolution crystal structures, we have revisited this problem. Apart from validating some of the earlier findings such as residue propensities, preferred location in the secondary structure, we have explored their spatial neighborhood preferences using the protein structure network PSN] approach developed in our lab. Finally, the structural and functional implications of the disallowed conformations are examined.

INDUCTION OF FOLDED STRUCTURES IN DESIGNED PEPTIDES USING CONFORMATIONALLY CONSTRAINED RESIDUES

Folding into compact globular structures, with well-defined modules of secondary structure, appears to be a characteristic of long polypeptide chains, with a specific patterning of coded amino acid residues along the length of sequence. Cooperative hydrogen bond driven secondary structure formation and solvent forces, which contribute favorably to the entropy of folding, by promoting compaction of the polymeric chain, have long been discussed as major determinants of the folding process. First principles design approaches, which use non-coded amino acids, employ an alternative structure directing strategy, by using amino acid residues which exhibit a strong conformational bias for specific regions of the Ramachandran map. This overview of ongoing studies in the authors' laboratory, attempts to explore the use of conformationally restricted amino acid residues in the design of peptides with well-defined secondary structures. Short peptides composed of 20 genetically coded amino acids usually exist in solution as an ensemble of equilibrating conformations. Apolar peptide sequences, which are readily soluble in organic solvents like chloroform and methanol, facilitate formation of structures which are predominately driven by intramolecular hydrogen bond formation. The choice of sequences containing residues with a limited range of conformational choices strongly favors formation of local turn structures, stabilized by short range intramolecular hydrogen bonds. Two residue beta-turns can nucleate either helical or hairpin folding, depending on the precise conformation of the turn segment Restriction of the conformational space available to amino acid residues is easily achieved by introduction of an additional alkyl group at the C alpha carbon atom or by side chain backbone cyclization, as in proline. Studies of synthetic sequences incorporating two prototype residues alpha-aminoisobutyric acid (Aib) and D-proline (DPro) illustrate the utility of the strategy in construction of helices and hairpins. Extensions to the design of conformationally switchable sequences and structurally defined hybrid peptides containing backbone homologated residues are also surveyed.

Hydrogen bonding, halogen bonding and lithium bonding: an atoms in molecules and natural bond orbital perspective towards conservation of total bond order, inter- and intra-molecular bonding

One hundred complexes have been investigated exhibiting D-X center dot center dot center dot A interactions, where X = H, Cl or Li and DX is the `X bond' donor and A is the acceptor. The optimized structures of all these complexes have been used to propose a generalized `Legon-Millen rule' for the angular geometry in all these interactions. A detailed Atoms in Molecules (AIM) theoretical analysis confirms an important conclusion, known in the literature: there is a strong correlation between the electron density at the X center dot center dot center dot A bond critical point (BCP) and the interaction energy for all these interactions. In addition, we show that extrapolation of the fitted line leads to the ionic bond for Li-bonding (electrostatic) while for hydrogen and chlorine bonding, it leads to the covalent bond. Further, we observe a strong correlation between the change in electron density at the D-X BCP and that at the X center dot center dot center dot A BCP, suggesting conservation of the bond order. The correlation found between penetration and electron density at BCP can be very useful for crystal structure analysis, which relies on arbitrary van der Waals radii for estimating penetration. Various criteria proposed for shared-and closed-shell interactions based on electron density topology have been tested for H/Cl/Li bonded complexes. Finally, using the natural bond orbital (NBO) analysis it is shown that the D-X bond weakens upon X bond formation, whether it is ionic (DLi) or covalent (DH/DCl) and the respective indices such as ionicity or covalent bond order decrease. Clearly, one can think of conservation of bond order that includes ionic and covalent contributions to both D-X and X center dot center dot center dot A bonds, for not only X = H/Cl/Li investigated here but also any atom involved in intermolecular bonding.

Thermoelectric properties of a Mn substituted synthetic tetrahedrite

Tetrahedrite compounds Cu12-xMnxSb4S13 (0 <= x <= 1.8) were prepared by solid state synthesis. A detailed crystal structure analysis of Cu10.6Mn1.4Sb4S13 was performed by single crystal X-ray diffraction (XRD) at 100, 200 and 300 K confirming the noncentrosymmetric structure (space group I (4) over bar 3m) of a tetrahedrite. The large atomic displacement parameter of the Cu2 atoms was described by splitting the 12e site into a partially and randomly occupied 24g site (Cu22) in addition to the regular 12e site (Cu21), suggesting a mix of dynamic and static off-plane Cu2 atom disorder. Rietveld powder XRD pattern and electron probe microanalysis revealed that all the Mn substituted samples showed a single tetrahedrite phase. The electrical resistivity increased with increasing Mn due to substitution of Mn2+ at the Cu1+ site. The positive Seebeck coefficient for all samples indicates that the dominant carriers are holes. Even though the thermal conductivity decreased as a function of increasing Mn, the thermoelectric figure of merit ZT decreased, because the decrease of the power factor is stronger than the decrease of the thermal conductivity. The maximum ZT = 0.76 at 623 K is obtained for Cu12Sb4S13. The coefficient of thermal expansion 13.5 +/- 0.1 x 10(-6) K-1 is obtained in the temperature range from 460 K to 670 K for Cu10.2Mn1.8Sb4S13. The Debye temperature, Theta(D) = 244 K for Cu10.2Mn1.8Sb4S13, was estimated from an evaluation of the elastic properties. The effective paramagnetic moment 7.45 mu(B)/f.u. for Cu10.2Mn1.8Sb4S13 is fairly consistent with a high spin 3d(5) ground state of Mn.

Directing peptide conformation with centrally positioned pre-organized dipeptide segments: studies of a 12-residue helix and beta-hairpin

Secondary structure formation in oligopeptides can be induced by short nucleating segments with a high propensity to form hydrogen bonded turn conformations. Type I/III turns facilitate helical folding while type II'/I' turns favour hairpin formation. This principle is experimentally verified by studies of two designed dodecapeptides, Boc-Val-Phe-Leu-Phe-Val-Aib-Aib-Val-Phe-Leu-Phe-Val-OMe 1 and Boc-Val-Phe-Leu-Phe-Val- (D) Pro- (L) Pro-Val-Phe-Leu-Phe-Val-OMe 2. The N- and C-terminal flanking pentapeptide sequences in both cases are identical. Peptide 1 adopts a largely alpha-helical conformation in crystals, with a small 3(10) helical segment at the N-terminus. The overall helical fold is maintained in methanol solution as evidenced by NMR studies. Peptide 2 adopts an antiparallel beta-hairpin conformation stabilized by 6 interstrand hydrogen bonds. Key nuclear Overhauser effects (NOEs) provide evidence for the antiparallel beta-hairpin structure. Aromatic proton chemical shifts provide a clear distinction between the conformation of peptides 1 (helical) and 2 (beta-hairpin). The proximity of facing aromatic residues positioned at non-hydrogen bonding positions in the hairpin results in extensively ring current shifted proton resonances in peptide 2.

Molecules support morphology: species status of South Indian populations of the widely distributed Hanuman langur

The taxonomy of the Hanuman langur (Semnopithecus spp.), a widely distributed Asian colobine monkey, has been in a flux for a long time due to much disagreement between various classification schemes. However, results from a recent field-based morphological study were consistent with Hill's (Ceylon J Sci 21:277-305, 1939) species level classification scheme. Here we tested the validity of S. hypoleucos and S. priam, the two South Indian species recognized by Hill. To this end, one mitochondrial and four nuclear markers were sequenced from over 72 non-invasive samples of Hanuman langurs and S. johnii collected from across India. The molecular data were subjected to various tree building methods. The nuclear data was also used in a Bayesian structure analysis and to determine the genealogical sorting index of each hypothesized species. Results from nuclear data suggest that the South Indian population of Hanuman langur consists of two units that correspond to the species recognized by Hill. However in the mitochondrial tree S. johnii and S. priam were polyphyletic probably due to retention of ancestral polymorphism and/or low levels of hybridization. Implications of these results on conservation of Hanuman langurs are also discussed.

Do we see what we should see? Describing non-covalent interactions in protein structures including precision

The power of X-ray crystal structure analysis as a technique is to `see where the atoms are'. The results are extensively used by a wide variety of research communities. However, this `seeing where the atoms are' can give a false sense of security unless the precision of the placement of the atoms has been taken into account. Indeed, the presentation of bond distances and angles to a false precision (i.e. to too many decimal places) is commonplace. This article has three themes. Firstly, a basis for a proper representation of protein crystal structure results is detailed and demonstrated with respect to analyses of Protein Data Bank entries. The basis for establishing the precision of placement of each atom in a protein crystal structure is non-trivial. Secondly, a knowledge base harnessing such a descriptor of precision is presented. It is applied here to the case of salt bridges, i.e. ion pairs, in protein structures; this is the most fundamental place to start with such structure-precision representations since salt bridges are one of the tenets of protein structure stability. Ion pairs also play a central role in protein oligomerization, molecular recognition of ligands and substrates, allosteric regulation, domain motion and alpha-helix capping. A new knowledge base, SBPS (Salt Bridges in Protein Structures), takes these structural precisions into account and is the first of its kind. The third theme of the article is to indicate natural extensions of the need for such a description of precision, such as those involving metalloproteins and the determination of the protonation states of ionizable amino acids. Overall, it is also noted that this work and these examples are also relevant to protein three-dimensional structure molecular graphics software.

Targeted Modifications in Adeno-Associated Virus Serotype 8 Capsid Improves Its Hepatic Gene Transfer Efficiency In Vivo

Recombinant adeno-associated virus vectors based on serotype 8 (AAV8) have shown significant promise for liver-directed gene therapy. However, to overcome the vector dose dependent immunotoxicity seen with AAV8 vectors, it is important to develop better AAV8 vectors that provide enhanced gene expression at significantly low vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal machinery, we modified specific serine/threonine kinase or ubiquitination targets on the AAV8 capsid to augment its transduction efficiency. Point mutations at specific serine (S)/threonine (T)/lysine (K) residues were introduced in the AAV8 capsid at the positions equivalent to that of the effective AAV2 mutants, generated successfully earlier. Extensive structure analysis was carried out subsequently to evaluate the structural equivalence between the two serotypes. scAAV8 vectors with the wild-type (WT) and each one of the S/T -> Alanine (A) or K-Arginine (R) mutant capsids were evaluated for their liver transduction efficiency in C57BL/6 mice in vivo. Two of the AAV8-S -> A mutants (S279A and S671A), and a K137R mutant vector, demonstrated significantly higher enhanced green fluorescent protein (EGFP) transcript levels (similar to 9- to 46-fold) in the liver compared to animals that received WT-AAV8 vectors alone. The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response, and a concomitant two-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector bio-distribution studies revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (106 vs. 7.7 vector copies/mouse diploid genome) when compared to WT-AAV8 vectors. To further study the utility of the K137R-AAV8 mutant in therapeutic gene transfer, we delivered human coagulation factor IX (h. FIX) under the control of liver-specific promoters (LP1 or hAAT) into C57BL/6 mice. The circulating levels of h. FIX: Ag were higher in all the K137R-AAV8 treated groups up to 8 weeks post-hepatic gene transfer. These studies demonstrate the feasibility of the use of this novel AAV8 vectors for potential gene therapy of hemophilia B.

Cell-Membrane-Mimicking Lipid-Coated Nanoparticles Confer Raman Enhancement to Membrane Proteins and Reveal Membrane-Attached Amyloid-beta Conformation

Identifying the structures of membrane bound proteins is critical to understanding their function in healthy and diseased states. We introduce a surface enhanced Raman spectroscopy technique which can determine the conformation of membrane-bound proteins, at low micromolar concentrations, and also in the presence of a substantial membrane-free fraction. Unlike conventional surface enhanced Raman spectroscopy, our approach does not require immobilization of molecules, as it uses spontaneous binding of proteins to lipid bilayer-encapsulated Ag nanoparticles. We apply this technique to probe membrane-attached oligomers of Amyloid-beta(40) (A beta(40)), whose conformation is keenly sought in the context of Alzheimer's disease. Isotope-shifts in the Raman spectra help us obtain secondary structure information at the level of individual residues. Our results show the presence of a beta-turn, flanked by two beta-sheet regions. We use solid-state NMR data to confirm the presence of the beta-sheets in these regions. In the membrane-attached oligomer, we find a strongly contrasting and near-orthogonal orientation of the backbone H-bonds compared to what is found in the mature, less-toxic A beta fibrils. Significantly, this allows a ``porin'' like beta-barrel structure, providing a structural basis for proposed mechanisms of A beta oligomer toxicity.

Mechano-fluorochromic Pt-II Luminogen and Its Cysteine Recognition

A new triphenylamine-based organometallic Pt-II luminogen (1) and its analogous organic compound (2) are reported. The molecules are decorated with aldehyde functionality to improve their photophysical properties by utilising donor-acceptor interactions. The single crystal X-ray structure analysis of PtII analogue 1 revealed that the neighbouring molecules were loosely organised by weak intermolecular C-H center dot center dot center dot pi interactions. Because of the twisted nature of the triphenylamine backbone the compounds showed aggregation-induced emission enhancement in THF/water mixture. Due to their loose crystal packing, upon application of external stimuli these luminogens exhibited mechano-fluorochromic behaviour. The crystalline forms of the compounds displayed a more superior emission efficiency than the grinded samples. Moreover, the compounds showed crystallization-induced emission enhancement (CIEE) and exhibited chemodosimetric response towards cysteine under physiological condition.

Covalent Postassembly Modification and Water Adsorption of Pd3 Self-Assembled Trinuclear Barrels

Three new ditopic imidazole ligands (2-4) were synthesized in high yields and characterized by various spectroscopic techniques. These ligands resulted in the formation of 3 + 6] self-assembled trinuclear barrels (5-7) in quantitative yields by stoichiometric combination of individual ligands and Pd(NO3)(2) in DMSO. All the three assemblies (5-7) were characterized by `H NMR and ESI-MS analysis, and subsequently, structures of the complexes 5 and 6 were confirmed by single-crystal X-ray diffraction studies. Structure analysis reveals the presence of NO3- counter anions in the intermolecular channels/pockets, which could potentially act as H-bonding sites between adsorbed water molecules within the channels. In fact, both the assemblies (5 and 6) showed water uptake (136.58, and 123.78 cm(3) g(-1), respectively) at ambient temperature under maximum allowable humidity. In addition, free aldehyde group present in the bridging ligand in complex 7 provides reactive site for postassembly modification. Herein, Knoevenagel condensation with Meldrum's acid was utilized under mild conditions by targeting aldehyde group appended in prefabricated complex 7 and transformed into a different complex (8) with altered functional group. Such postassembly functionalization enables incorporation of a new functional group without disrupting the integrity of the trifacial structure.

Cytokine control of neuronal phenotype

Diffusible proteins regulate neural development at a variety of stages. Using a novel neuronal culture assay, I have identified several cytokines that regulate the expression of neurotransmitters and neuropeptides in sympathetic neurons. These cytokines fall into two families. The first group is termed the neuropoietic cytokines, while including CDF/LIF, CNTF, OSM and GPA, induces expression of the same set of neuropeptide mRNAs in cultured sympathetic neurons. These four factors not only exhibit similar biological activities; they also share a predicted secondary structure and bind to a signal-transducing receptor subunit in common with IL-6 and IL-11. The latter two cytokines display a weaker activity in this assay. In addition, I find that several members of the TGF-β superfamily, activin A, BMP-2, and BMP-6, have a selective overlap with the neuropoietic family in the spectrum of neuropeptides that these cytokines induce in sympathetic neurons. Different patterns of neuropeptides induced by the TGF-β family members, however, demonstrate that the activities of these cytokines are distinct from those of the neuropoietic family. Another 30 cytokines are without detectable effect in this neuronal assay.

Activin A induces a set of neurotransmitters and neuropeptides that is somewhat similar to the phenotype of sympathetic neurons innervating sweat glands in rat footpads. In situ hybridization and RNase protection were carried out to test whether activins were involved in the phenotypic transition when sympathetic neurons contact sweat glands. I find that activin mRNA is present in both cholinergic and noradrenergic targets. Moreover, homogenates of footpads do not contain activin-like activity in the neuronal assay in vitro. Taken together, these data do not support activins as the best candidates for the sweat gland factor.

Several novel factors that regulate neuropeptide expression exist in heart cell conditioned medium. I attempted to purify these factors in collaboration with Dr. Jane Talvenheimo. Our results suggest that these factors are sensitive to the storage conditions used. Several modifications of purification strategy are discussed.