Abstract
Bacillus subtilis possesses three homologous small cold shock proteins (CSPs; CspB, CspC, CspD, sequence identity >72%). They share a similar beta-sheet structure, as shown by circular dichroism, and have a very low conformational stability, with CspC being the least stable. Similar to CspB, CspC and CspD unfold and refold extremely fast in a N <==> U two-state reaction with average lifetimes of only 100-150 ms for the native state and 1-6 ms for the unfolded states at 25 degreesC. As a consequence of their low stability and low kinetic protection against unfolding, all three cold shock proteins are rapidly degraded by proteases in vitro. Analysis of the CSP stabilities in vivo by pulse-chase experiments revealed that CspB and CspD are stable during logarithmic growth at 37 degreesC as well as after cold shock. The cellular half-life of CspC is shortened at 37 degreesC, but under cold shock conditions CspC becomes stable. The proteolytic susceptibility of the CSPs in vitro was strongly reduced in the presence of a nucleic acid ligand, suggesting that the observed stabilization of CSPs in vivo is mediated by binding to their substrate mRNA at 37 degreesC and, in particular, under cold shock conditions.
Highlights
Bacillus subtilis possesses three homologous small cold shock proteins (CSPs; CspB, CspC, CspD, sequence identity >72%)
CspC was significantly stabilized after a cold shock. These findings suggest that CSPs are complexed with a nucleic acid ligand in the cell, under cold shock conditions as well as at 37 °C, and are thereby protected against proteolytic attack
We showed that CSPs are stabilized in the presence of a limiting amount of a nucleic acid ligand in vitro, and we propose that in vivo CSPs exist in a tight complex with their biological ligand and are thereby stabilized
Summary
(Received for publication, July 28, 1998, and in revised form, November 24, 1998). Thomas Schindler‡§¶, Peter L. Bacillus subtilis possesses three homologous small cold shock proteins (CSPs; CspB, CspC, CspD, sequence identity >72%). They share a similar -sheet structure, as shown by circular dichroism, and have a very low conformational stability, with CspC being the least stable. The proteolytic susceptibility of the CSPs in vitro was strongly reduced in the presence of a nucleic acid ligand, suggesting that the observed stabilization of CSPs in vivo is mediated by binding to their substrate mRNA at 37 °C and, in particular, under cold shock conditions. We investigated the stability and the folding kinetics of CspC and CspD of B. subtilis These two cold shock proteins resemble CspB in their rapid unfolding and refolding and in their low thermodynamic stability. These findings suggest that CSPs are complexed with a nucleic acid ligand in the cell, under cold shock conditions as well as at 37 °C, and are thereby protected against proteolytic attack
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