TY - JOUR
T1 - The initial step of the thermal unfolding of 3-isopropylmalate dehydrogenase detected by the temperature-jump Laue method
AU - Hori, Tetsuya
AU - Moriyama, Hideaki
AU - Kawaguchi, Jitsutaro
AU - Hayashi-Iwasaki, Yoko
AU - Oshima, Tairo
AU - Tanaka, Nobuo
PY - 2000
Y1 - 2000
N2 - 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 β-turn and a loop located between the two domains of the enzyme. A mutant, 2T2M6T-E110P/S111G/S113E, having amino acid replacements in this β-turn region with the corresponding residues of the thermophilic enzyme, showed greater stability than the original chimera (increase of T(m) 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 β-turn region.
AB - 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 β-turn and a loop located between the two domains of the enzyme. A mutant, 2T2M6T-E110P/S111G/S113E, having amino acid replacements in this β-turn region with the corresponding residues of the thermophilic enzyme, showed greater stability than the original chimera (increase of T(m) 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 β-turn region.
KW - 3-Isopropylmalate dehydrogenase
KW - Crystal structure
KW - Temperature-jump Laue method
KW - Thermal unfolding
KW - Thermostability
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U2 - 10.1093/protein/13.8.527
DO - 10.1093/protein/13.8.527
M3 - Article
C2 - 10964981
AN - SCOPUS:0033824928
VL - 13
SP - 527
EP - 533
JO - Protein Engineering, Design and Selection
JF - Protein Engineering, Design and Selection
SN - 1741-0126
IS - 8
ER -