A simplified recipe for assigning amide NMR signals using combinatorial (14)N amino acid inverse-labeling.

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A simplified recipe for assigning amide NMR signals using combinatorial (14)N amino acid inverse-labeling.

A simplified recipe for assigning amide NMR signals using combinatorial (14)N amino acid inverse-labeling.

J Struct Funct Genomics. 2011 Aug 25;

Authors: Hiroaki H, Umetsu Y, Nabeshima YI, Hoshi M, Kohda D

Abstract
Assignment of backbone amide proton resonances is one of the most time-consuming stages of any protein NMR study when the protein samples behave non-ideally. A robust and convenient NMR procedure for analyzing spectra of marginal-to-low quality is helpful for high-throughput structure determination. The (14)N selective- and inverse-labeling method is a candidate solution. Here, we present a simplified protocol for assigning protein backbone amide NMR signals. When (14)N inversely labeled residues are present in a protein, their backbone NH cross peaks vanish from the protein's (1)H-(15)N HSQC spectrum, and thus, their chemical shifts can be readily identified by a process of elimination. Some metabolically related amino acids, for example, Ile, Leu, and Val, cannot be individually incorporated but can be inversely labeled together. We optimized and simplified the protocol and M9-based medium formula for the (14)N selective- and inverse-labeling method without any additives. Our approach should be cost-effective, because the method could be additively applied stepwise, even when the proteins of interest were found to be non-ideal.


PMID: 21866395 [PubMed - as supplied by publisher]



Source: PubMed