Functional regions of rice heat shock protein, Oshsp16.9, required for conferring thermotolerance in Escherichia coli.
Rice (Oryza sativa) class I low-molecular mass (LMM) heat shock protein (HSP), Oshsp16.9, has been shown to be able to confer thermotolerance in Escherichia coli. To define the regions for this intriguing property, deletion mutants of this hsp have been constructed and overexpressed in E. coli XL1-blue cells after isopropyl beta-D-thioglactopyranoside induction. The deletion of amino acid residues 30 through 36 (PATSDND) in the N-terminal domain or 73 through 78 (EEGNVL) in the consensus II domain of Oshsp16.9 led to the loss of chaperone activities and also rendered the E. coli incapable of surviving at 47.5 degrees C. To further investigate the function of these two domains, we determined the light scattering changes of Oshsp16.9 mutant proteins at 320 nm under heat treatment either by themselves or in the presence of a thermosensitive enzyme, citrate synthase. It was observed that regions of amino acid residues 30 through 36 and 73 through 78 were responsible for stability of Oshsp16.9 and its interactions with other unfolded protein substrates, such as citrate synthase. Studies of two-point mutants of Oshsp16.9, GST-N74E73K and GST-N74E74K, indicate that amino acid residues 73 and 74 are an important part of the substrate-binding site of Oshsp16.9. Non-denaturing gel analysis of purified Oshsp16.9 revealed that oligomerization of Oshsp16.9 was necessary but not sufficient for its chaperone activity.
Yeh
Ching-Hui
CH
Department of Botany, National Taiwan University, Taipei, Taiwan, Republic of China.
Chen
Yih-Ming
YM
Lin
Chu-Yung
CY
D001665:Binding Sites / Q000235:genetics; D002950:Citrate (si)-Synthase / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006358:Hot Temperature; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008027:Light; D009154:Mutation; D012275:Oryza / Q000378:metabolism*; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D006360:Heat-Shock Proteins; C108681:Oshsp16.9 protein, Oryza sativa; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D007544:Isopropyl Thiogalactoside; D002950:Citrate (si)-Synthase; D005982:Glutathione Transferase