Selective 15N-labeling of the side-chain amide groups of asparagine and glutamine for applications in paramagnetic NMR spectroscopy
Abstract
The side-chain amide groups of asparagine and glutamine play important roles in stabilizing the structural fold of proteins, participating in hydrogen-bonding networks and protein interactions. Selective 15N-labeling of side-chain amides, however, can be a challenge due to enzyme-catalyzed exchange of amide groups during protein synthesis. In the present study, we developed an efficient way of selectively labeling the side chains of asparagine, or asparagine and glutamine residues with 15NH2. Using the biosynthesis pathway of tryptophan, a protocol was also established for simultaneous selective 15N-labeling of the side-chain NH groups of asparagine, glutamine, and tryptophan. In combination with site-specific tagging of the target protein with a lanthanide ion, we show that selective detection of 15N-labeled side-chains of asparagine and glutamine allows determination of magnetic susceptibility anisotropy tensors based exclusively on pseudocontact shifts of amide side-chain protons.
Source: Journal of Biomolecular NMR