The initial step of the thermal unfolding of 3-isopropylmalate dehydrogenase detected by the temperature-jump Laue method.

Tetsuya Hori1,2, Hideaki Moriyama3,4, and IR spectroscopy (Phillips et al., 1995; Reinstadler et al., Jitsutaro Kawaguchi1,5, Yoko Hayashi-Iwasaki6,7, 1996). Many attempts have also been made to determine in Tairo Oshima6 and Nobuo Tanaka1 detail the structures of intermediate species, for instance, by NMR (Balbach et al., 1995; Eliezer et al., 1998; Udgaonkar 1Graduate School of Bioscience and Biotechnology, Tokyo Institute of and Baldwin, 1988), X-ray diffraction (Gillbert et al., 1982) Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8501, 2RIKEN and molecular dynamics simulations (reviewed in Daggett and Harima Institute, Kouto 1–1–1, Mikazuki-cho, Sayo-gun, Hyogo 679-5148, 3Experimental Facilities Division, Japan Synchrotron Radiation Research Levitt, 1994). However, it is still difficult to elucidate the Institute, SPring-8, Kouto 1–1–1, Mikazuki-cho, Sayo-gun, Hyogo 679-5198 structural dynamics of protein folding/unfolding processes at and 6Department of Molecular Biology, Tokyo University of Pharmacy and the atomic level, especially for relatively large proteins, to Life Science, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan which NMR analysis is not applicable. 5Present address: Centre for Genome Research, University of Edinburgh, We have developed an X-ray crystallographic structural Kings Buildings, West Mains Road, Edinburgh EH9 3JQ, UK analysis technique coupled with a temperature jump (T-jump) 7Present address: Department of Bioengineering, Nagaoka University of induced by laser irradiation to determine the structural change Technology, Kamitomioka-cho 1603-1, Nagaoka, Niigata 940-2188, Japan of proteins induced by the T-jump, at the atomic level. 4To whom correspondence should be addressed. Gillbert et al. (1982) previously attempted to identify the E-mail: aki5@sp8sun.spring8.or.jp conformational changes of crystalline ribonuclease A induced by a stepwise temperature increase, by following the changes A temperature-jump (T-jump) time-resolved X-ray crystalin X-ray diffraction and resonance Raman signals. Their results lographic technique using the Laue method was developed suggested the existence of an initial denaturation site in the to detect small, localized structural changes of proteins in protein from which all further denaturation propagates and crystals exposed to a temperature increase induced by laser they observed several structural changes as crystals were irradiation. In a chimeric protein between thermophilic and mesophilic 3-isopropylmalate dehydrogenases warmed close to the denaturation temperature. However, loss (2T2M6T), the initial structural change upon T-jump to a of diffraction and radiation damage to the crystals prevented denaturing temperature (~90°C) was found to be localized detailed structural analysis at higher temperatures. We bypassed at a region which includes a β-turn and a loop located these problems by utilizing polychromatic Laue crystallobetween the two domains of the enzyme. A mutant, graphic methods, which permit an increased rate of data 2T2M6T-E110P/S111G/S113E, having amino acid replacecollection, so that the structural details of even an unstable ments in this β-turn region with the corresponding residues intermediate can be visualized. This technique has been applied of the thermophilic enzyme, showed greater stability than to reveal the crystal structures of several transient catalytic the original chimera (increase of Tm by ~10°C) and no Tintermediates (Schlichting et al., 1990; Bolduc et al., 1995; jump-induced structural change in this region was detected Stoddard, 1998; Stoddard et al., 1998). In the present T-jump by our method. These results indicate that thermal Laue method, a protein crystal was heated extremely quickly unfolding of the original chimeric enzyme, 2T2M6T, is by YAG laser radiation and Laue images were recorded within triggered in this β-turn region. 40 ms. The T-jump generated by the laser was estimated to