A systematic mutagenesis-driven strategy for site-resolved NMR studies of supramolecular assemblies
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Obtaining sequence-specific assignments remains a major bottleneck in solution NMR investigations of supramolecular structure, dynamics and interactions. Here we demonstrate that resonance assignment of methyl probes in high molecular weight protein assemblies can be efficiently achieved by combining fast NMR experiments, residue-type-specific isotope-labeling and automated site-directed mutagenesis. The utility of this general and straightforward strategy is demonstrated through the characterization of intermolecular interactions involving a 468-kDa multimeric aminopeptidase, PhTET2.
KeywordsNMR spectroscopy High molecular weight proteins Assignment Site-specific mutagenesis Methyl-group labeling
We thank Dr. P. Gans, R. Rasia, P. Schanda and J.-P. Simorre for critical reading of the manuscript, the Partnership for Structural Biology for access to High-Field NMR, RoBioMol and Isotopic Labeling platforms. CA acknowledges funding from CONACYT; TV, BF and JB acknowledge funding from an interdisciplinary Program of the CNRS and Agence Nationale de la Recherche (ANR-09-PIRIBio-445583 & ANR-07-BLAN-0085).
- Fischer M, Kloiber K, Hausler J, Ledolter K, Konrat R, Schmid W (2007) Synthesis of a 13C-methyl-group-labeled methionine precursor as a useful tool for simplifying protein structural analysis by NMR spectroscopy. Chem Biochem 8:610–612Google Scholar
- Keller R (2004) The computer aided resonance assignment tutorial: CANTINA. Verlag, GoldauGoogle Scholar