A systematic mutagenesis-driven strategy for site-resolved NMR studies of supramolecular assemblies
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
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