構造生物 Vol.2 No1
1996年4月発行

Thermostability of proteins: Knowing the primary mechanism, so what?


Hideaki Moriyama and Nobuo Tanaka

Department of Lifescience, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuda, Midori-Ku, Yokohama 227 Japan

We have been studied thermostability of protein with using 3-isopropylmalate dehydrogenase isolated from a thermophile, Thermus thermophilus. Studies were based on destruction of native by means of site-directed mutagenesis andlor formation of hybrid protein between thermophile and mesophile. Crystallographic investigations of those mimic molecules indicated unfavorable conformation, which was local but was significant, made the protein heat-sensitive. The unstable clues were local repulsions, elongated hydrogen bonds, high energetic torsion angles and so on. Those particular obstacles were able to remove by the reversion from mimic type to thermophile. Although we can see the primary mechanism, we did not give any answer against a question: "Why the protein is thermostable?" Structure biology may contribute the investigation on the thermostability of protein through precise and real-shape structure analysis. In the system of 3- isopropylmalate dehydrogenase, high water content in the crystal made the structure analysis incomplete. The resulted structures have high basal level of temperature factor due to the 78% of completeness of reflection data collection, 6% of measurement error. Another problem is temperature at the data collection. Those data had been collected at the room temperature, however the optimal temperature of the present enzyme is more than 60。C. To overcome those problem we employ the crystal structure analysis in different temperature utilizing cryogenic data collection and temperature jump coupled with the Laue method. Cryogenic structure analysis of native thermostable enzyme gave us phase transition point at near 150K, however mimic cS82R did not. This result indicated that the multiple conformation of the mimic protein at the cryogenic temperature. Laser irradiation coupled with Laue structure analysis gave us temperature response movement of hydrophilic residues on the molecular surface. Those preliminary results are good forecast, however we have a lot of things to do.


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