Residue-specific insights into (2x)72 kDa tryptophan synthase obtained from fast-MAS 1H-detected solid-state NMR

Klein, A.; Rovó, P.; Sakhrani, V. V.; Wang, Y.; Holmes, J.; Liu, V.; Skowronek, P.; Kukuk, L.; Vasa, S. K.; Güntert, P.; Mueller, L. J.; Linser, R.

doi:10.1101/2021.05.12.443859

bioRxiv · 10.1101/2021.05.12.443859

Residue-specific insights into (2x)72 kDa tryptophan synthase obtained from fast-MAS 1H-detected solid-state NMR

Klein, A.·Rovó, P.·Sakhrani, V. V.·Wang, Y.·Holmes, J.·Liu, V.·Skowronek, P.·Kukuk, L.·Vasa, S. K.·Güntert, P.·Mueller, L. J.·Linser, R.

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Abstract

Solid-state NMR has emerged as a potent technique in structural biology, suitable for the study of fibrillar, micro-crystalline, and membrane proteins. Recent developments in fast-magic-angle-spinning and proton-detected methods have enabled detailed insights into structure and dynamics, but molecular-weight limitations for the asymmetric part of target proteins have remained at ~30-40 kDa. Here we employ solid-state NMR for atom-specific characterization of the 72 kDa (asymmetric unit) microcrystalline protein tryptophan synthase, an important target in pharmacology and biotechnology, chemical-shift assignments of which we obtain via higher-dimensionality, 4D and 5D solid-state NMR experiments. The assignments for the first time provide comprehensive data for assessment of side chain chemical properties involved in the catalytic turnover, and, in conjunction with first-principles calculations, precise determination of thermodynamic and kinetic parameters is demonstrated for the essential acid-base catalytic residue {beta}K87. The insights provided by this study expand by nearly a factor of two the size limitations widely accepted for NMR today, demonstrating the applicability of solid-state NMR to systems that have been thought to be out of reach due to their complexity.

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Klein, A., Rovó, P., Sakhrani, V. V., Wang, Y., Holmes, J., Liu, V., Skowronek, P., Kukuk, L., Vasa, S. K., Güntert, P., Mueller, L. J., Linser, R.. 2021-05-14. Residue-specific insights into (2x)72 kDa tryptophan synthase obtained from fast-MAS 1H-detected solid-state NMR. https://doi.org/10.1101/2021.05.12.443859

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