Nanosecond-Timescale Dynamics and Conformational Heterogeneity in Human GCK Regulation and Disease |
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Institution: | 1. Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida;2. Department of Biological Science, Florida State University, Tallahassee, Florida;3. Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida;4. Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota;5. Department of Chemistry, University of Minnesota, Minneapolis, Minnesota;6. Center for Molecular Modeling, Center for Information Technology, National Institutes of Health, Bethesda, Maryland |
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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. |
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