Human glucokinase (GCK) is the prototypic example of an emerging class of proteins with allosteric-like behavior that originates from intrinsic polypeptide dynamics. High-resolution NMR investigations of GCK have elucidated millisecond-timescale dynamics underlying allostery. In contrast, faster motions have remained underexplored, hindering the development of a comprehensive model of cooperativity. Here, we map nanosecond-timescale dynamics and structural heterogeneity in GCK using a combination of unnatural amino acid incorporation, time-resolved fluorescence, and 19F nuclear magnetic resonance spectroscopy. We find that a probe inserted within the enzyme's intrinsically disordered loop samples multiple conformations in the unliganded state. Glucose binding and disease-associated mutations that suppress cooperativity alter the number and/or relative population of these states. Together, the nanosecond kinetics characterized here and the millisecond motions known to be essential for cooperativity provide a dynamical framework with which we address the origins of cooperativity and the mechanism of activated, hyperinsulinemia-associated, noncooperative variants.
Bibliographical noteFunding Information:
This work was supported by grants from the National Institutes of Health ( GM115388 and GM133843 ) to B.G.M. and ( GM100310 ) to G.V.. The Center for Information Technology is by the Intramural Research Program of the National Institutes of Health .
© 2020 Biophysical Society
PubMed: MeSH publication types
- Journal Article
- Research Support, N.I.H., Extramural
- Research Support, N.I.H., Intramural