| Exposure of organisms to sub-lethal temperatures leads to the synthesis of sets of proteins called heat shock proteins (HSPs). There are five classes of heat shock proteins based on their subunit molecular weights. This study has focused on the smallest heat shock proteins, sHSPs, from a hyperthermophilic archaeon, Pyrococcus furiosus. Most sHSPs share amino acid sequence similarity and chaperone functions with α-crystallin, a major vertebrate eye lens protein. Small HSPs have subunit molecular weight of up to 40 kDa and most of them form large molecular weight complexes ranging in size from 200 to 800 kDa. In P. furiosus, the protein is not synthesized at 95°C but the synthesis of sHSP is induced by heat shock (105°C). PfusHSP can restrain Escherichia coli cell-free extracts as well as a mesophilic bovine glutamate dehydrogenase from precipitation at 105 and 50°C, respectively. However, the Pfu-sHSP did not prevent the loss of glutamate dehydrogenase activity suggesting that the Pfu-sHSP may function only to prevent denatured proteins from aggregation. In addition, Pfu-sHSP can enhance E. coli survivability of 6.5-fold at 50°C and also promote growth of E. coli at 45 and 47.5°C.; The functions of Pfu-sHSP have been investigated by constructing mutants based on the two available crystal structures of sHSPs. Similar to most sHSPs, Pfu-sHSP is polydisperse. The amino terminal domain is not involved in subunit assembly. However, the carboxy terminal domain appears to be significantly involved in subunit assembly as the mutant lacking the 12-amino acid carboxy terminal domain disrupted complex formation. In addition, this mutant was unable to form large complexes and appears to lack in vivo function in E. coli. The Pfu-sHSP also contributes to biotechnology applications, as it can improve the stability of DNA polymerase and reduce the DNA polymerase levels required in PCR reactions. |
