The long term objective of this research is to understand the molecular mechanisms which enable thermophilic microorganisms to survive and grow in conditions of extreme heat. This proposal is focused on elucidating the subtle structural features of the glyceraldehyde-3-phosphate dehydrogenase (GPDH) that contribute to remarkably different heat stabilities of the enzyme from two phylogenetically related thermophiles. The extremely thermostable GPDH from the obligate thermophile B. stearothermophilus and the unusually thermolabile form of the same enzyme from the facultative thermophile B. coagulans together provide a unique model system for the study of the bases of protein thermostability. The power of the model lies in the fact that in spite of their enormous differences in heat stability, the two forms of the enzyme exhibit an unusual degree of protein homology. Utilizing molecular and classical genetic procedures to probe this model, the following studies will be carried out. l) The cloned genes (gap) that specify the two forms of the enzyme will be sequenced by the dideoxy chain termination method. The amino acid sequence for that portion of the B. coagulans enzyme for which the sequence is not known will be deduced from the nucleotide sequence. 2) Hybrid gap genes will be created both by in vitro genetic engineering procedures and by in vivo recombination in E. coli. 3) Mutants of B. stearothermophilus with temperature sensitive mutations in the gap gene and of B. coagulans with temperature-resistant mutations in the gene will also be isolated. 4) Hybrid gap genes and the gap genes of those mutants that synthesize GPDHs with altered heat stability will be cloned and sequenced. 5) Amino acid sequences deduced from the nucleotide sequences of the hybrid and mutant gap genes will then be compared to those of the wild-type GPDHs, in order to identify regions of the polypeptides which contribute to the stability. 6) Finally, site-directed mutagenesis will be utilized to pinpoint those amino acids that are essential for the heat stability of the GPDH protein. These studies will contribute to the general knowledge of protein structure by defining the molecular reasons responsible for stability or lack of stability in enzymes. Ultimately such knowledge will lead to a greater understanding of the alterations in protein turnover and degenerative structural changes that occur in the aging process.
