Atomic-resolution chemical characterization of (2x)72-kDa tryptophan synthase via four- and five-dimensional 1H-detected solid-state NMR

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 119; no. 4; p. 1
• Main Authors: Klein, Alexander, Rovó, Petra, Sakhrani, Varun V, Wang, Yangyang, Holmes, Jacob B, Liu, Viktoriia, Skowronek, Patricia, Kukuk, Laura, Vasa, Suresh K, Güntert, Peter, Mueller, Leonard J, Linser, Rasmus
• Format: Journal Article
• Language: English
• Published: Washington National Academy of Sciences 25.01.2022
• Subjects: Amino acids; Backbone; Biological Sciences; Chemical properties; Crystallography; Electron microscopy; First principles; NMR; Nuclear magnetic resonance; Proteins; Solid state; Tryptophan; Tryptophan synthase; X-ray crystallography
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2114690119

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.