Biomedical subjects

Satish Raina

Publications and source records attributed to Satish Raina.

5 recordsLinked to original sources

PubMed2006

Fatty acyl benzamido antibacterials based on inhibition of DnaK-catalyzed protein folding.

We have reported that the hsp70 chaperone DnaK from Escherichia coli might assist protein folding by catalyzing the cis/trans isomerization of secondary amide peptide bonds in unfolded or partially folded proteins. In this study a series of fatty acylated benzamido inhibitors of the cis/trans isomerase activity of DnaK was developed and tested for antibacterial effects in E. coli MC4100 cells. N(alpha)-[Tetradecanoyl-(4-aminomethylbenzoyl)]-l-asparagine is the most effective antibacterial with a minimal inhibitory concentration of 100 +/- 20 microg/ml. The compounds were shown to compete with fluorophore-labeled sigma(32)-derived peptide for the peptide binding site of DnaK and to increase the fraction of aggregated proteins in heat-shocked bacteria. Despite its inability to serve as a folding helper in vivo a DnaK-inhibitor complex was still able to sequester an unfolded protein in vitro. Structure activity relationships revealed a distinct dependence of DnaK-assisted refolding of luciferase on the fatty acyl chain length, whereas the minimal inhibitory concentration was most sensitive to the structural nature of the benzamido core. We conclude that the isomerase activity of DnaK is a major survival factor in the heat shock response of bacteria and that small molecule inhibitors can lead to functional inactivation of DnaK and thus will display antibacterial activity.

Anti-Bacterial Agents↗

PubMed2004

Crystal structures of the DsbG disulfide isomerase reveal an unstable disulfide.

Dsb proteins control the formation and rearrangement of disulfide bonds during the folding of secreted and membrane proteins in bacteria. DsbG, a member of this family, has disulfide bond isomerase and chaperone activity. Here, we present two crystal structures of DsbG at 1.7and 2.0-A resolution that are meant to represent the reduced and oxidized forms, respectively. The oxidized structure, however, reveals a mixture of both redox forms, suggesting that oxidized DsbG is less stable than the reduced form. This trait would contribute to DsbG isomerase activity, which requires that the active-site Cys residues are kept reduced, regardless of the highly oxidative environment of the periplasm. We propose that a Thr residue that is conserved in the cis-Pro loop of DsbG and DsbC but not found in other Dsb proteins could play a role in this process. Also, the structure of DsbG reveals an unanticipated and surprising feature that may help define its specific role in oxidative protein folding. Thus, the dimensions and surface features of DsbG show a very large and charged binding surface that is consistent with interaction with globular protein substrates having charged surfaces. This finding suggests that, rather than catalyzing disulfide rearrangement in unfolded substrates, DsbG may preferentially act later in the folding process to catalyze disulfide rearrangement in folded or partially folded proteins.

Amino Acid Sequence↗

PubMed2003

Dehydration converts DsbG crystal diffraction from low to high resolution.

Diffraction quality crystals are essential for crystallographic studies of protein structure, and the production of poorly diffracting crystals is often regarded as a dead end in the process. Here we show a dramatic improvement of poorly diffracting DsbG crystals allowing high-resolution diffraction data measurement. Before dehydration, the crystals are fragile and the diffraction pattern is streaky, extending to 10 A resolution. After dehydration, there is a spectacular improvement, with the diffraction pattern extending to 2 A resolution. This and other recent results show that dehydration is a simple, rapid, and inexpensive approach to convert poor quality crystals into diffraction quality crystals.

Crystallography, X-Ray↗

PubMed2003

Phosphorylation-mediated regulation of heat shock response in Escherichia coli.

Escherichia coli has two heat shock regulons under the transcriptional control of Esigma32 and EsigmaE RNA polymerases. These polymerases control the expression of genes, the products of which are needed for correct folding of proteins in the cytoplasm and the extracytoplasm respectively. In this study, we report that mutations in a tyrosine phosphatase-encoding gene led to decreased activity of these heat shock regulons. The activity of the tyrosine phosphatase is presumably co-ordinated with that of a cognate kinase. We show here that mutants deleted for the phosphatase-encoding gene accumulate phosphorylated RpoH. We find that RpoH is phosphorylated at amino acid position 260, which is located in the conserved region 4.2, and that this phosphorylation event attenuates RpoH activity as a sigma factor. The rpoH Tyr-260Ala mutation confers a temperature-sensitive phenotype that leads to an altered heat shock response. Additionally, we show that the antisigma factor RseA is phosphorylated at the N-terminally located Tyr-38 and that this phosphorylation presumably alters its binding affinity towards sigmaE.

Escherichia coli↗

PubMed2002

Interaction of the conserved region 4.2 of sigma(E) with the RseA anti-sigma factor.

Esigma(E) RNA polymerase transcribes a regulon of folding factors for the bacterial envelope and is induced by physical and chemical stresses. The RseA anti-sigma factor inhibits the activity of Esigma(E) RNA polymerase. It is shown here that the N-terminal portion of sigma(E), residues 1-153, binds core RNA polymerase. RseA interacts with residues 154-191 of sigma(E), a site that is homologous to region 4, the sigma factor binding site for promoter DNA. Mutations that reduce transcription of Esigma(E) RNA polymerase map to sigma(E) residues 178, 181, and 183. Variant sigma(E) proteins with amino acid substitutions at residues 178, 181, or 183 do not associate with RseA. A regulatory mechanism is proposed whereby RseA binds to a C-terminal peptide of sigma(E) and inhibits the transcription of Esigma(E) RNA polymerase by blocking promoter recognition.

Cloning, Molecular↗