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Functional characterization of Xenopus small heat shock protein, Hsp30C: the carboxyl end is required for stability and chaperone activity.
Fernando P
,
Heikkila JJ
.
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Small heat shock proteins protect cells from stress presumably by acting as molecular chaperones. Here we report on the functional characterization of a developmentally regulated, heat-inducible member of the Xenopus small heat shock protein family, Hsp30C. An expression vector containing the open reading frame of the Hsp30C gene was expressed in Escherichia coli. These bacterial cells displayed greater thermoresistance than wild type or plasmid-containing cells. Purified recombinant protein, 30C, was recovered as multimeric complexes which inhibited heat-induced aggregation of either citrate synthase or luciferase as determined by light scattering assays. Additionally, 30C attenuated but did not reverse heat-induced inactivation of enzyme activity. In contrast to an N-terminal deletion mutant, removal of the last 25 amino acids from the C-terminal end of 30C severely impaired its chaperone activity. Furthermore, heat-treated concentrated solutions of the C-terminal mutant formed nonfunctional complexes and precipitated from solution. Immunoblot and gel filtration analysis indicated that 30C binds with and maintains the solubility of luciferase preventing it from forming heat-induced aggregates. Coimmunoprecipitation experiments suggested that the carboxyl region is necessary for 30C to interact with target proteins. These results clearly indicate a molecular chaperone role for Xenopus Hsp30C and provide evidence that its activity requires the carboxyl terminal region.
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