STRUCTURE ANALYSIS BY MEANS OF TEMPERATURE JUMP COUPLED WITH THE LAUE METHOD. Hideaki Moriyama, Noriyuki Igarashi, Akira Ikezaki, Nobuo Tanaka. Department of Life Science, Faculty of Biotechnology and Bioscience, Tokyo Institute of Technology, 4259 Nagatsuta, MIDORI-ku, Yokohama, 227 Japan

Temperature jump coupled with the Laue diffraction method has been postulated to analyze crystal structures at a high temperature in a short time period. This method may permit an opportunity of collection of diffraction data prior to destruction of crystal lattice. Rapid increase of crystal temperature was achieved by a laser impact with an infrared-ray irradiation.

The first applications of temperature jump experiments were applied on the structure analysis of 3-isopropylmalate dehydrogenase that isolated from a thermophile (1). The temperature jump device has been developed by a cooperation between Rigaku Co. Ltd. and us. The diffraction experiments in a mode of temperature jump coupled with the Laue method were performed at Beam Line 18B of Photon Factory at Institute of High Energy Physics in Tsukuba, Japan. A crystal was mounted as a usual manner then laser was input for a period of time and white ray was incident by an automated and/or manually linked temperature jump controller. The exposure time was 10 mS. The collected diffraction images on large imagine plates, 400 mm x 800 mm, were processed by the in PF-in-house programs including index, intlaue, lpcor, and lauenorm. The process had been done up to 2.25 Å resolution over the wavelength range between 0.85 and 2.35 Å. The processed data gave R factor, Imean from all measurements for the reflection, at 0.094 in the both data set.

The structure of 3-isopropylmalate dehydrogenase was directly refined using the intact and t-jump Laue data, those refinements gave crystallographic R factor of 0.18 and 0.19, respectively. The temperature factors for the intact and t-jump structures were 22 Å square. The structural r.m.s.d was 0.72 Å and the major differences were found in surface-hydrophilic residues.

1. M. Sakurai, K. Onodera, H. Moriyama, T. Oshima & N. Tanaka, Protein Engineering, 8, 763-767 (1995).