| Autor: |
Sternisha, Shawn M., Whittington, A. Carl, Martinez Fiesco, Juliana A., Porter, Carol, McCray, Malcolm M., Logan, Timothy, Olivieri, Cristina, Veglia, Gianluigi, Steinbach, Peter J., Miller, Brian G. |
| Zdroj: |
Biophysical Journal; 20240101, Issue: Preprints |
| Abstrakt: |
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. |
| Databáze: |
Supplemental Index |
| Externí odkaz: |
|
