Nanosecond-Timescale Dynamics and Conformational Heterogeneity in Human GCK Regulation and Disease

Shawn M. Sternisha, A. Carl Whittington, Juliana A. Martinez Fiesco, Carol Porter, Malcolm M. McCray, Timothy Logan, Cristina Olivieri, Gianluigi Veglia, Peter J. Steinbach, Brian G. Miller

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1109-1118
Number of pages10
JournalBiophysical journal
Volume118
Issue number5
DOIs
StatePublished - Mar 10 2020

Bibliographical note

Funding 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 .

Publisher Copyright:
© 2020 Biophysical Society

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural

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