Temperature-Resolved Cryo-EM Uncovers Structural Bases of Temperature-Dependent Enzyme Functions.
J Am Chem Soc
; 141(51): 19983-19987, 2019 12 26.
Article
in En
| MEDLINE
| ID: mdl-31829582
ABSTRACT
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 °C, and show that structures of both the Mg2+ form (KARI2Mg2+) and its ternary complex (KARI2Mg2+NADHinhibitor) 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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Temperature
/
Ketol-Acid Reductoisomerase
Language:
En
Journal:
J Am Chem Soc
Year:
2019
Type:
Article
Affiliation country:
Taiwan