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Facilitating Unambiguous NMR Assignment and Enabling Higher Probe Density by Selective Labeling of all Methyl Containing Amino Acids

Proudfoot, Andrew and Frank, Andreas and Ruggiu, Fiorella and Lingel, Andreas (2016) Facilitating Unambiguous NMR Assignment and Enabling Higher Probe Density by Selective Labeling of all Methyl Containing Amino Acids. Journal of Biomolecular NMR.

Abstract

The deuteration of proteins and selective labeling of side chain methyl groups has greatly enhanced the molecular weight range of protein and protein complexes which can be studied using solution NMR spectroscopy. Protocols for the selective labeling of all six methyl group containing amino acids individually are available, however to date, only a maximum combination of five amino acids have been labeled simultaneously. Here, we describe a new methodology for the simultaneous, selective labeling of all six methyl containing amino acids using the 115 kDa homohexameric enzyme CoaD from E. coli as a model system. The utility of the labeling protocol is demonstrated by efficiently and unambiguously assigning all methyl groups in the enzymatic active site using a single 4D 13C-resolved HMQC-NOESY-HMQC experiment, in conjunction with a crystal structure. Furthermore, the six fold labeled protein was employed to characterize the interaction between the substrate analogue (R)-pantetheine and CoaD by chemical shift perturbations, demonstrating the benefit of the increased probe density.

Item Type: Article
Date Deposited: 29 Apr 2016 23:45
Last Modified: 29 Apr 2016 23:45
URI: https://oak.novartis.com/id/eprint/28353

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