, Volume 38, Issue 1, pp 89-98
Date: 28 Mar 2007

1-13C amino acid selective labeling in a 2H15N background for NMR studies of large proteins

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Abstract

Isotope labeling by residue type (LBRT) has long been an important tool for resonance assignments at the limit where other approaches, such as triple-resonance experiments or NOESY methods do not succeed in yielding complete assignments. While LBRT has become less important for small proteins it can be the method of last resort for completing assignments of the most challenging protein systems. Here we present an approach where LBRT is achieved by adding protonated 14N amino acids that are 13C labeled at the carbonyl position to a medium for uniform deuteration and 15N labeling. This has three important benefits over conventional 15N LBRT in a deuterated back ground: (1) selective TROSY-HNCO cross peaks can be observed with high sensitivity for amino-acid pairs connected by the labeling, and the amide proton of the residue following the 13C labeled amino acid is very sharp since its alpha position is deuterated, (2) the 13C label at the carbonyl position is less prone to scrambling than the 15N at the α-amino position, and (3) the peaks for the 1-13C labeled amino acids can be identified easily from the large intensity reduction in the 1H-15N TROSY-HSQC spectrum for some residues that do not significantly scramble nitrogens, such as alanine and tyrosine. This approach is cost effective and has been successfully applied to proteins larger than 40 kDa.