Protein Engineering, Vol. 13, No. 8, 527-533,
August 2000
© 2000 Oxford University Press
The initial step of the thermal unfolding of 3-isopropylmalate dehydrogenase detected by the temperature-jump Laue method
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
A temperature-jump (T-jump) time-resolved X-ray crystallographic technique using the Laue method was developed to detect small, localized structural changes of proteins in crystals exposed to a temperature increase induced by laser irradiation. In a chimeric protein between thermophilic and mesophilic 3-isopropylmalate dehydrogenases (2T2M6T), the initial structural change upon T-jump to a denaturing temperature (~90°C) was found to be localized at a region which includes a ß-turn and a loop located between the two domains of the enzyme. A mutant, 2T2M6T-E110P/S111G/S113E, having amino acid replacements in this ß-turn region with the corresponding residues of the thermophilic enzyme, showed greater stability than the original chimera (increase of Tm by ~10°C) and no T-jump-induced structural change in this region was detected by our method. These results indicate that thermal unfolding of the original chimeric enzyme, 2T2M6T, is triggered in this ß-turn region.