Abstract
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.
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Acknowledgments
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).
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C. Amero and M. Asuncion Dura contributed equally to this work.
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Amero, C., Asunción Durá, M., Noirclerc-Savoye, M. et al. A systematic mutagenesis-driven strategy for site-resolved NMR studies of supramolecular assemblies. J Biomol NMR 50, 229–236 (2011). https://doi.org/10.1007/s10858-011-9513-5
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DOI: https://doi.org/10.1007/s10858-011-9513-5