Modelling multiple disulphide loop containing polypeptides by random conformation generation. The test cases of α-conotoxin GI and edothelin I

A general procedure for arriving at 3-D models of disulphiderich olypeptide systems based on the covalent cross-link constraints has been developed. The procedure, which has been coded as a computer program, RANMOD, assigns a large number of random, permitted backbone conformations to the polypeptide and identifies stereochemically acceptable structures as plausible models based on strainless disulphide bridge modelling. Disulphide bond modelling is performed using the procedure MODIP developed earlier, in connection with the choice of suitable sites where disulphide bonds could be engineered in proteins (Sowdhamini,R., Srinivasan,N., Shoichet,B., Santi,D.V., Ramakrishnan,C. and Balaram,P. (1989) Protein Engng, 3, 95-103). The method RANMOD has been tested on small disulphide loops and the structures compared against preferred backbone conformations derived from an analysis of putative disulphide subdatabase and model calculations. RANMOD has been applied to disulphiderich peptides and found to give rise to several stereochemically acceptable structures. The results obtained on the modelling of two test cases, a-conotoxin GI and endothelin I, are presented. Available NMR data suggest that such small systems exhibit conformational heterogeneity in solution. Hence, this approach for obtaining several distinct models is particularly attractive for the study of conformational excursions.

On the involvement of intramolecular protein disulfide in the irreversible inactivation of 3-hydroxy-3-methylglutaryl-CoA reductase by diallyl disulfide

Treatment with diallyl disulfide, a constituent of garlic oil, irreversibly inactivated microsomal and a soluble 50 kDa form of HMG-CoA reductase. No radioactivity was found to be protein-bound on treating the soluble enzyme with [35S]diallyl disulfide, indicating the absence of the mixed disulfide of the type allyl-S-S-protein. SDS-PAGE and Western blot analyses of the diallyl-disulfide-treated protein showed no traces of the dimer of the type protein-S-S-protein, but clearly indicated BME-reversible increased mobility, as expected of an intramolecular protein disulfide. The sulfhydryl groups, as measured by alkylation with iodo[2-14C]acetic acid, were found to decrease in the diallyl-disulfide-treated enzyme protein. Tryptic peptide analysis also gave support for the possible presence of disulfide-containing peptides in such a protein. It appears that diallyl disulfide inactivated HMG-CoA reductase by forming an internal protein disulfide that became inaccessible for reduction by DTT, and thereby retaining the inactive state of the enzyme.

Geometry of proline-containing alpha-helices in proteins

Crystal structure analysis of proline-containing alpha-helices in proteins has been carried out. High resolution crystal structures were selected from the Protein Data Bank. Apart from the standard internal parameters, some parameters which are specifically related to the bend in the helix due to proline have been developed and analyzed. Finally the position and nature of these helices and their interactions with the rest of the protein have been analyzed.

Bioinorganic and medicinal chemistry: aspects of gold(I)-protein complexes

Gold(I)-based drugs have been used successfully for the treatment of rheumatoid arthritis (RA) for several years. Although the exact mechanism of action of these gold(I) drugs for RA has not been clearly established, the interaction of these compounds with mammalian enzymes has been extensively studied. In this paper, we describe the interaction of therapeutic gold(I) compounds with mammalian proteins that contain cysteine (Cys) and selenocysteine (Sec) residues. Owing to the higher affinity of gold(I) towards sulfur and selenium, gold(I) drugs rapidly react with the activated cysteine or selenocysteine residues of the enzymes to form protein-gold(I)-thiolate or protein-gold(I)-selenolate complexes. The formation of stable gold(I)-thiolate/selenolate complexes generally lead to inhibition of the enzyme activity. The gold-thiolate/selenolate complexes undergo extensive ligand exchange reactions with other nucleophiles and such ligand exchange reactions alter the inhibitory effects of gold(I) complexes. Therefore, the effect of gold(I) compounds on the enzymatic activity of cysteine-or selenocysteine-containing proteins may play important roles in RA. The interaction of gold(I) compounds with different enzymes and the biochemical mechanism underlying the inhibition of enzymatic activities may have broad medicinal implications for the treatment of RA.

Cloning and characterization of a cluster of genes coding for 5S rRNA and three tRNAs from Azospirillum lipoferum

A genomic library was constructed from a HindIII digest of Azospirillum lipoferum chromosomal DNA in the HindIII site of pUC19. From the library, a clone, pALH64, which showed strong hybridization with 3' end labeled A. lipoferum total tRNAs and which contains a 2.9 kb insert was isolated and restriction map of the insert established. The nucleotide sequence of a 490 bp HindIII-HincII subfragment containing a cluster of genes coding for 5S rRNA, tRNA(Val)(UAC), tRNA(Thr)(UGA) and tRNA(Lys)(UUU) has been determined. The gene organization is 5S rRNA (115 bp), spacer (10 bp), tRNA(Val) (76 bp), spacer (3 bp), tRNA(Thr) (76 bp), spacer (7 bp) and tRNA(Lys) (76 bp). Hybridization experiments using A. lipoferum total tRNAs and 5S rRNA with the cloned DNA probes revealed that all three tRNA genes and the 5S rRNA gene are expressed in vivo in the bacterial cells.

Inhibition of nuclear protein import by a monoclonal antibody against a novel class of nuclear pore proteins

Nuclear import of proteins is mediated by the nuclear pore complexes in the nuclear envelope and requires the presence of a nuclear localization signal (NLS) on the karyophilic protein. In this paper, we describe studies with a monoclonal antibody, Mab E2, which recognizes a class of nuclear pore proteins of 60-76 kDa with a common phosphorylated epitope on rat nuclear envelopes. The Mab Ea-reactive proteins fractionated with the relatively insoluble pore complex-containing component of the envelope and gave a finely punctate pattern of nuclear staining in immunofluorescence assays. The antibody did not bind to any cytosolic proteins. Mab E2 inhibited the interaction of a simian virus 40 large T antigen NLS peptide with a specific 60-kDa NLS-binding protein from rat nuclear envelopes in photoaffinity labeling experiments. The antibody blocked the nuclear import of NLS-albumin conjugates in an in vitro nuclear transport assay with digitonin-permeabilized cells, but did not affect passive diffusion of a small nonnuclear protein, lysozyme, across the pore. Mab E2 may inhibit protein transport by directly interacting with the 60-kDa NLS-binding protein, thereby blocking signal-mediated nuclear import across the nuclear pore complex. (C) 1994 Academic Press, Inc.

Interaction of ecoP15I DNA methyltransferase with oligonucleotides containing the asymmetric sequence 5?-CAGCAG-3

EcoP15I DNA methyltransferase (Mtase) recognizes the asymmeteric sequence CAGCAG and catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the second adenine residue. We have investigated the DNA binding properties of EcoP15I DNA Mtase using gel mobility shift assays. EcoP15I DNA Mtase binds approximately threefold more tightly to DNA containing its recognition sequence, CAGCAG, than to non-specific sequences in the absence or presence of cofactors. Interestingly, in the presence of ATP the discrimination between specific and non-specific sequences increases significantly. These results suggest for the first time a role for ATP in DNA recognition by type III restriction-modification enzymes. In addition, we have shown that bromodeoxyuridine-containing oligonucleotides form complexes with EcoP15I DNA Mtase that are crosslinked upon irradiation. More importantly, we have shown that the crosslink site is at the site of DNA binding, since it can be suppressed by an excess of unmodified oligonucleotide. EcoP15I DNA Mtase exhibited Michaelis-Menten kinetics with both unmodified and bromodeoxyuridine-substituted DNA, with a higher specificity constant for the latter. Furthermore, gel mobility shift assays showed that proteolyzed EcoP15I DNA Mtase formed a specific complex with DNA, which had similar mobility as the native protein-DNA complex. Taken together these results form the basis fora detailed structure-function analysis of EcoP15I DNA Mtase.

Diffusion path during internal displacement reactions in multi-component oxides: Reaction between Fe and (Co,Mg)TiO3 solid solution at 1273 K

The reaction between Fe foil and a disc of ilmenite solid solution (Co-0.48 Ni-0.52) TiO3 was studied at 1273 K. At the metal/oxide interface, the displacement reaction, Fe + (Co,Mg)TiO3 = Co + (Fe,Mg)TiO3 occurs, resulting in an ilmenite solid solution containing three divalent cations. Ferrous ions diffuse into the oxide solid solution and cause the precipitation of Co-Fe alloy as discrete particles inside the oxide matrix. The morphology of the product layer was characterized by SEM. Only two phases, alloy and ilmenite, were detected in the reaction zone. This suggests that the local flux condition imposed by ilmenite stoichiometry (Co + Fe + Mg):Ti = 1:1] was satisfied during the reactive diffusion: (J(Co) + J(Fe) + J(Mg)) = J(Ti). The composition of the alloy and the oxide was determined using EPMA as a function of distance in the direction of diffusion. Although Mg does not participate in the displacement reaction, its composition in the ilmenite phase was found to be position dependent inside the reaction zone. The up-hill diffusion of inert Mg is caused by the development of chemical potential gradients as a result of displacement reaction. The evolution of composition gradients inside the reaction zone and the diffusion path in a ternary composition diagram of the system CoTiO3-FeTiO3-MgTiO3 are discussed. (C) 2010 Elsevier B.V. All rights reserved.

In vitro regeneration from immature cotyledon explant and protein profile changes during organogenesis in groundnut (Arachis hypogaea L.)

Complete plants were regenerated from in vitro cultured immature cotyledon segments of groundnut (Arachis hypogaea L. cv. TMV-7) by organogenesis. Callus cultures were best Initiated from immature cotyledon segments on MS (Murashige and Skoog) salts containing B5 vitamins supplemented with indole-3-acetic acid (IAA) and alpha -naphthalene acetic acid (NAA; 4.0 mg L-1) and kinetin (KIN; 0.5 L-1). Calluses were transferred to a medium containing KIN (2.0 mg L-1) and IAA and NAA (0.5 mg L-1) for shoot Initiation. The regenerated shoots were transferred to a medium containing Indole-3-butyric acid (IBA; 2.0 mg L-1) and KIN (0.2 mg L-1) for developing roots. In vitro produced plantlets developed sucessfully, matured, and set seed. The protein profiles [sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE)] of callus, callus with shoot, and callus with shoot and root showed differences.

Metal-billed and hollow carbon nanotubes obtained by the decomposition of metal-containing free precursor molecules

Vapor-phase pyrolysis of Fe(CO)(5) in the presence of another carbon source such as CO or Ca He yields iron-filled or hollow nanotubes depending on the relative concentration of the carbon source. Essentially single-walled nanotubes are obtained when the C6H6/Fe(CO)(5) ratio is high. Pyrolysis of metallocenes yields metal-filled nanotubes and hollow nanotubes are obtained when metallocenes are pyrolyzed along with benzene. Metal-decorated nanotubes are also obtained by this method.

Nitrogen-containing carbon nanotubes

Carbon nanotubes containing small amounts of nitrogen are produced by the pyrolysis of aza-aromatics such as pyridine, methylpyrimidine and triazine over cobalt nanoparticles in an Ar atmosphere; good yields of such nanotubes are obtained by carrying out the pyrolysis of a mixture of pyridine and Fe(CO)(5) in flowing Ar + H-2.

Quantum clustering and network analysis of MD simulation trajectories to probe the conformational ensembles of protein-ligand interactions

In this article, we present a novel application of a quantum clustering (QC) technique to objectively cluster the conformations, sampled by molecular dynamics simulations performed on different ligand bound structures of the protein. We further portray each conformational population in terms of dynamically stable network parameters which beautifully capture the ligand induced variations in the ensemble in atomistic detail. The conformational populations thus identified by the QC method and verified by network parameters are evaluated for different ligand bound states of the protein pyrrolysyl-tRNA synthetase (DhPylRS) from D. hafniense. The ligand/environment induced re-distribution of protein conformational ensembles forms the basis for understanding several important biological phenomena such as allostery and enzyme catalysis. The atomistic level characterization of each population in the conformational ensemble in terms of the re-orchestrated networks of amino acids is a challenging problem, especially when the changes are minimal at the backbone level. Here we demonstrate that the QC method is sensitive to such subtle changes and is able to cluster MD snapshots which are similar at the side-chain interaction level. Although we have applied these methods on simulation trajectories of a modest time scale (20 ns each), we emphasize that our methodology provides a general approach towards an objective clustering of large-scale MD simulation data and may be applied to probe multistate equilibria at higher time scales, and to problems related to protein folding for any protein or protein-protein/RNA/DNA complex of interest with a known structure.

Synthesis of single-walled carbon nanotubes using binary (Fe, Co, Ni) alloy nanoparticles prepared in situ by the reduction of oxide solid solutions

Passing a H-2-CH4 mixture over oxide spinels containing two transition elements as in Mg0.8MyMz'Al2O4 (M, M' = Fe, Co or Ni, y + z = 0.2) at 1070 degrees C produces small alloy nanoparticles which enable the formation of carbon nanotubes. Surface area measurements are found to be useful for assessing the yield and quality of the nanotubes. Good-quality single-walled nanotubes (SWNTs) have been obtained in high yields with the FeCo alloy nanoparticles, as evidenced by transmission electron microscope images and surface area measurements. The diameter of the SWNTs is in the 0.8-5 nm range, and the multiwalled nanotubes, found occasionally, possess very few graphite layers. (C) 1999 Elsevier Science B.V. All rights reserved.

FE analysis of piezo-laminate composites under thermal loads

A shear flexible 4-noded finite element formulation, having five mechanical degrees of freedom per node, is presented for modeling the dynamic as well as the static thermal response of laminated composites containing distributed piezoelectric layers. This element has been developed to have one electrical degree of freedom per piezoelectric layer. The mass, stiffness and thermo-electro-mechanical coupling effects on the actuator and sensor layers have been considered. Numerical studies have been conducted to investigate both the sensory and active responses on piezoelectric composite beam and plate structures. It is. concluded that both the thermal and pyroelectric effects are important and need to be considered in the precision distributed control of intelligent structures.

PDB Goodies - a web-based GUI to manipulate the Protein Data Bank file

PDB Goodies is a web-based graphical user interface (GUI) to manipulate the Protein Data Bank file containing the three-dimensional atomic coordinates of protein structures. The program also allows users to save the manipulated three-dimensional atomic coordinate file on their local client system. These fragments are used in various stages of structure elucidation and analysis. This software is incorporated with all the three-dimensional protein structures available in the Protein Data Bank, which presently holds approximately 18 000 structures. In addition, this program works on a three-dimensional atomic coordinate file (Protein Data Bank format) uploaded from the client machine. The program is written using CGI/PERL scripts and is platform independent. The program PDB Goodies can be accessed over the World Wide Web at http:// 144.16.71.11/pdbgoodies/.