期刊
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
卷 141, 期 51, 页码 19983-19987出版社
AMER CHEMICAL SOC
DOI: 10.1021/jacs.9b10687
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资金
- Academia Sinica [AS-CFII-108-110]
- Taiwan Protein Project [AS-KPQ-105-TPP]
- Academia Sinica
- Taiwan Ministry of Science and Technology [MOST 108-2113-M-008-008]
- Taiwan Protein Project
Protein functions are temperature-dependent, but protein structures are usually solved at a single (often low) temperature because of limitations on the conditions of crystal growth or protein vitrification. Here we demonstrate the feasibility of solving cryo-EM structures of proteins vitrified at high temperatures, solve 12 structures of an archaeal ketol-acid reductoisomerase (KARI) vitrified at 4-70 degrees C, and show that structures of both the Mg2+ form (KARI:2Mg(2+)) and its ternary complex (KARI:2Mg(2+):NADH:inhibitor) are temperature-dependent in correlation with the temperature dependence of enzyme activity. Furthermore, structural analyses led to dissection of the induced-fit mechanism into ligand-induced and temperature-induced effects and to capture of temperature-resolved intermediates of the temperature-induced conformational change. The results also suggest that it is preferable to solve cryo-EM structures of protein complexes at functional temperatures. These studies should greatly expand the landscapes of protein structure-function relationships and enhance the mechanistic analysis of enzymatic functions.
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