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How wide is the window opened by high-resolution relaxometry on the internal dynamics of proteins in solution?

ABSTRACT: The dynamics of molecules in solution is usually quantified by the determination of timescale-specific amplitudes of motions. High-resolution nuclear magnetic resonance (NMR) relaxometry experiments-where the sample is transferred to low fields for longitudinal (T₁) relaxation, and back to high field for detection with residue-specific resolution-seeks to increase the ability to distinguish the contributions from motion on timescales slower than a few nanoseconds. However, tumbling of a molecule in solution masks some of these motions. Therefore, we investigate to what extent relaxometry improves timescale resolution, using the "detector" analysis of dynamics. Here, we demonstrate improvements in the characterization of internal dynamics of methyl-bearing side chains by carbon-13 relaxometry in the small protein ubiquitin. We show that relaxometry data leads to better information about nanosecond motions as compared to high-field relaxation data only. Our calculations show that gains from relaxometry are greater with increasing correlation time of rotational diffusion.

SUBMITTER: Smith AA

PROVIDER: S-EPMC8018934 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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How wide is the window opened by high-resolution relaxometry on the internal dynamics of proteins in solution?

Smith Albert A AA Bolik-Coulon Nicolas N Ernst Matthias M Meier Beat H BH Ferrage Fabien F

Journal of biomolecular NMR 20210323 2-3

The dynamics of molecules in solution is usually quantified by the determination of timescale-specific amplitudes of motions. High-resolution nuclear magnetic resonance (NMR) relaxometry experiments-where the sample is transferred to low fields for longitudinal (T<sub>1</sub>) relaxation, and back to high field for detection with residue-specific resolution-seeks to increase the ability to distinguish the contributions from motion on timescales slower than a few nanoseconds. However, tumbling of ...[more]

PMID: 33759077

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Project description:The remarkable power and specificity of enzyme catalysis rely on the dynamic alignment of the enzyme, substrates, and cofactors, yet the role of dynamics has usually been approached from the perspective of the protein. We have been using an underappreciated NMR technique, subtesla high-resolution field cycling 31P NMR relaxometry, to investigate the dynamics of the enzyme-bound substrates and cofactor on guanosine-5'-monophosphate reductase (GMPR). GMPR forms two dead end, yet catalytically competent, complexes that mimic distinct steps in the catalytic cycle: E·IMP·NADP+ undergoes a partial hydride transfer reaction, while E·GMP·NADP+ undergoes a partial deamination reaction. A different cofactor conformation is required for each partial reaction. Here we report the effects of mutations designed to perturb cofactor conformation and ammonia binding with the goal of identifying the structural features that contribute to the distinct dynamic signatures of the hydride transfer and deamination complexes. These experiments suggest that Asp129 is a central cog in a dynamic network required for both hydride transfer and deamination. In contrast, Lys77 modulates the conformation and mobility of substrates and cofactors in a reaction-specific manner. Thr105 and Tyr318 are part of a deamination-specific dynamic network that includes the 2'-OH of GMP. These residues have comparatively little effect on the dynamic properties of the hydride transfer complex. These results further illustrate the potential of high-resolution field cycling NMR relaxometry for the investigation of ligand dynamics. In addition, exchange experiments indicate that NH3/NH4+ has a high affinity for the deamination complex but a low affinity for the hydride transfer complex, suggesting that the movement of ammonia may gate the cofactor conformational change. Collectively, these experiments reinforce the view that the enzyme, substrates, and cofactor are linked in intricate, reaction-specific, dynamic networks and demonstrate that distal portions of the substrates and cofactors are critical features in these networks.

Dataset Information

How wide is the window opened by high-resolution relaxometry on the internal dynamics of proteins in solution?

Publications

How wide is the window opened by high-resolution relaxometry on the internal dynamics of proteins in solution?

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