Atomic-resolution chemical characterization of (2x)72-kDa tryptophan synthase via four- and five-dimensional 1H-detected solid-state NMR
Edited by Robert Tycko, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD; received August 12, 2021; accepted December 13, 2021
Significance
The atomic-level understanding of protein function and enzyme catalysis requires site-specific information on chemical properties such as protonation and hybridization states and chemical exchange equilibria. This information is encoded in NMR chemical shifts, which serve as important complementary information to structural data from other experimental techniques or structure prediction algorithms. This study demonstrates that comprehensive chemical-shift assignments are achievable for large and highly complex proteins, offering insights into chemical structure and dynamics. The access to the active-site chemistry in the 144-kDa (72-kDa asymmetric unit) enzyme tryptophan synthase demonstrated here extends the elucidation of chemical properties to a member of an important class of enzymes of interest in pharmacology and biotechnology.
Abstract
NMR chemical shifts provide detailed information on the chemical properties of molecules, thereby complementing structural data from techniques like X-ray crystallography and electron microscopy. Detailed analysis of protein NMR data, however, often hinges on comprehensive, site-specific assignment of backbone resonances, which becomes a bottleneck for molecular weights beyond 40 to 45 kDa. Here, we show that assignments for the (2x)72-kDa protein tryptophan synthase (665 amino acids per asymmetric unit) can be achieved via higher-dimensional, proton-detected, solid-state NMR using a single, 1-mg, uniformly labeled, microcrystalline sample. This framework grants access to atom-specific characterization of chemical properties and relaxation for the backbone and side chains, including those residues important for the catalytic turnover. Combined with first-principles calculations, the chemical shifts in the β-subunit active site suggest a connection between active-site chemistry, the electrostatic environment, and catalytically important dynamics of the portal to the β-subunit from solution.
Data Availability
Acknowledgments
We thank the group of W. Koźmiński and Jan Stanek for helpful discussions about Signal Separation Algorithm (SSA) and related processing scripts. R.L. was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – 27112786, 325871075, and the Emmy Noether program. This study was funded by the DFG under Germany’s Excellence Strategy – EXC 2033 – 390677874 – RESOLV and EXC-114 – 24286268 – CiPS-M (Ruhr Explores SOLVation and Center for integrated Protein Science Munich, respectively). Financial support is also acknowledged from US NSF Grant CHE1710671 and NIH Grants GM097569 and GM137008 given to L.J.M. We acknowledge the computing time provided on the Linux High-Performance Computing cluster at Technical University Dortmund (LiDO3), partially funded via the Large-Scale Equipment Initiative by the DFG as Project 271512359. Additional computations were performed using the computer clusters and data storage resources of the University of California, Riverside High-Performance Computer Cluster, funded by grants from NSF (MRI-1429826) and NIH (S10OD016290).
Supporting Information
Materials/Methods, Supplementary Text, Tables, Figures, and/or References
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Copyright © 2022 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).
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Received: August 12, 2021
Accepted: December 13, 2021
Published online: January 20, 2022
Published in issue: January 25, 2022
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Acknowledgments
We thank the group of W. Koźmiński and Jan Stanek for helpful discussions about Signal Separation Algorithm (SSA) and related processing scripts. R.L. was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – 27112786, 325871075, and the Emmy Noether program. This study was funded by the DFG under Germany’s Excellence Strategy – EXC 2033 – 390677874 – RESOLV and EXC-114 – 24286268 – CiPS-M (Ruhr Explores SOLVation and Center for integrated Protein Science Munich, respectively). Financial support is also acknowledged from US NSF Grant CHE1710671 and NIH Grants GM097569 and GM137008 given to L.J.M. We acknowledge the computing time provided on the Linux High-Performance Computing cluster at Technical University Dortmund (LiDO3), partially funded via the Large-Scale Equipment Initiative by the DFG as Project 271512359. Additional computations were performed using the computer clusters and data storage resources of the University of California, Riverside High-Performance Computer Cluster, funded by grants from NSF (MRI-1429826) and NIH (S10OD016290).
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The authors declare no competing interest.
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Atomic-resolution chemical characterization of (2x)72-kDa tryptophan synthase via four- and five-dimensional 1H-detected solid-state NMR, Proc. Natl. Acad. Sci. U.S.A.
119 (4) e2114690119,
https://doi.org/10.1073/pnas.2114690119
(2022).
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