In the standard method of transformation of Escherichia coli with extraneous DNA, cells are made competent for DNA uptake by incubating in ice-cold 100 mM CaCl(2). Analysis of the whole protein profile of CaCl(2)-treated E. coli cells by the techniques of one- and two-dimensional gel electrophoresis, MALDI-MS and immunoprecipitation revealed overproduction of outer membrane proteins OmpC, OmpA and heat-shock protein GroEL. In parity, transformation efficiency of E. coli ompC mutant by plasmid pUC19 DNA was found to be about 40 % lower than that of the wild type strain. Moreover, in E. coli cells containing groEL-bearing plasmid, induction of GroEL caused simultaneous overproduction of OmpC. On the other hand, less OmpC was synthesized in E. coli groEL mutant compared to its wild type counterpart, by CaCl(2)-shock. From these results it can be suggested that in the process of CaCl(2)-mediated generation of competence, the heat-shock chaperone GroEL has specific role in DNA entry into the cell, possibly through the overproduced OmpC and OmpA porins.
Edited by Stuart Ferguson
Keywords:Stability of r 32 Physiological temperature DnaK GroEL Chaperone network E. coli a b s t r a c tThe stability of heat-shock transcription factor r 32 in Escherichia coli has long been known to be modulated only by its own transcribed chaperone DnaK. Very few reports suggest a role for another heat-shock chaperone, GroEL, for maintenance of cellular r 32 level. The present study demonstrates in vivo physical association between GroEL and r 32 in E. coli at physiological temperature. This study further reveals that neither DnaK nor GroEL singly can modulate r 32 stability in vivo; there is an ordered network between them, where GroEL acts upstream of DnaK.
E. coli small heat shock proteins IbpA and IbpB (inclusion body binding proteins A and B) are known to act as holding chaperones on denaturing, aggregate-prone proteins. But, there is no clear understanding about which of the IbpA and IbpB has more holdase activity and how the holdase activity of one was influenced by the presence of the other. This study was conducted to resolve the questions, using some uncommon physical techniques like dynamic light scattering, micro-viscometry and atomic force microscopy in addition to the common techniques of spectrophotometry and spectrofluorimetry. The holdase activity was investigated on the heat-denatured L-lactate dehydrogenase (LDH) of rabbit muscle. LDH was found to be deactivated completely without any aggregation at 52°C and with transient aggregation at 60°C; molecular dynamics simulation also revealed that at 52°C, denaturation occurred only at the active site of LDH. When LDH was allowed to be deactivated in the presence of IbpA, IbpB or (IbpA + IbpB), partial inhibition of i) denaturation at 52°C and ii) aggregation at 60°C were observed. The results further demonstrated that the holdase activity of IbpB was higher than that of IbpA and their combined effect was higher than their individual one.
The heat shock response mechanism is a very vital biochemical process and is mainly controlled by σ32 protein. The function of σ32
is temperature dependent and at lower temperatures σ32 is inactivated by its interactions with DnaK. This interaction is completely
abolished above 42°C till date no molecular details of the interactions are available. In the present scenario, an attempt has been
made to analyze first the predicted structure of σ32 obtained by comparative modeling techniques and then to study the interactions
between σ32 and DnaK. From this molecular modeling study we could specifically identify the binding sites of the interactions of
σ32 with DnaK which will enlighten the mechanism of regulation of its activity and stability by DnaK. Our study provides the idea
for future mutational experiments in order to find out the possible roles of the amino acids of region2 and region3 of σ32 in stability
as well as in binding with DnaK.
It has been recently reported through classical molecular dynamics simulations, that potassium ions have lower binding affinity for glutamate residues than water, leading to destabilization of the helical conformations of the peptide. In contrast, sodium ions have much stronger affinity for glutamate groups than for water, strongly stabilizing the helical conformations of the peptide. On the other hand, recent CD and UVRR experiments found that both ions: sodium and potassium, have the same effect, inducing just a very small stabilization on the helical conformations of the polypeptide for concentrations greater than 1M. In this work, we investigate the controversy presented above by performing classical molecular dynamics simulations of the poly-l-glutamate immersed in pure water, sodium chloride and potassium chloride. We present alpha helical contents in each solvent and give a quantitative estimation of how the barrier between alpha helix and unfolded states is affected by the presence of the ions.
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