Atomic-resolution chemical characterization of (2x)72-kDa tryptophan synthase via four- and five-dimensional
| Autor: | Alexander, Klein, Petra, Rovó, Varun V, Sakhrani, Yangyang, Wang, Jacob B, Holmes, Viktoriia, Liu, Patricia, Skowronek, Laura, Kukuk, Suresh K, Vasa, Peter, Güntert, Leonard J, Mueller, Rasmus, Linser |
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| Rok vydání: | 2021 |
| Předmět: |
Models
Molecular Protein Conformation NMR crystallography Biological Sciences Crystallography X-Ray Molecular Weight Biophysics and Computational Biology tautomerism PLP-dependent enzymes Tryptophan Synthase solid-state NMR tryptophan synthase Protein Multimerization Nuclear Magnetic Resonance Biomolecular Protein Binding |
| Zdroj: | Proceedings of the National Academy of Sciences of the United States of America |
| ISSN: | 1091-6490 |
| Popis: | 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. 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. |
| Databáze: | OpenAIRE |
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