The initial step of the thermal unfolding of 3-isopropylmalate dehydrogenase detected by the temperature-jump Laue method |
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| Authors: | Hori Tetsuya; Moriyama Hideaki; Kawaguchi Jitsutaro; Hayashi-Iwasaki Yoko; Oshima Tairo; Tanaka Nobuo |
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| Affiliation: | 1 Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8501,
2 RIKEN Harima Institute, Kouto 1–1–1, Mikazuki-cho, Sayo-gun, Hyogo 679-5148,
3 Experimental Facilities Division, Japan Synchrotron Radiation Research Institute, SPring-8, Kouto 1–1–1, Mikazuki-cho, Sayo-gun, Hyogo 679-5198 and
6 Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan |
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| Abstract: | A temperature-jump (T-jump) time-resolved X-ray crystallographictechnique using the Laue method was developed to detect small,localized structural changes of proteins in crystals exposedto a temperature increase induced by laser irradiation. In achimeric protein between thermophilic and mesophilic 3-isopropylmalatedehydrogenases (2T2M6T), the initial structural change uponT-jump to a denaturing temperature (~90°C) was found tobe localized at a region which includes a ß-turn and aloop located between the two domains of the enzyme. A mutant,2T2M6T-E110P/S111G/S113E, having amino acid replacements inthis ß-turn region with the corresponding residues ofthe thermophilic enzyme, showed greater stability than the originalchimera (increase of Tm by ~10°C) and no T-jump-inducedstructural change in this region was detected by our method.These results indicate that thermal unfolding of the originalchimeric enzyme, 2T2M6T, is triggered in this ß-turn region. |
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