TROSY of side-chain amides in large proteins

摘要

By using the mixed solvent of 50% H2O/50% D2O and employing deuterium decoupling, TROSY experiments exclusively detect NMR signals from semideuterated isotopomers of carboxamide groups with high sensitivities for proteins with molecular weights up to 80 kDa. This isotopomer-selective strategy extends TROSY experiments from exclusively detecting backbone to both backbone and side-chain amides, particularly in large proteins. Because of differences in both TROSY effect and dynamics between N-15-H-E{D-Z} and N-15-H-Z{D-E} isotopomers of the same carboxamide, the N-15 transverse magnetization of the latter relaxes significantly faster than that of the former, which provides a direct and reliable stereospecific distinction between the two configurations. The TROSY effects on the N-15-H-E{D-Z} isotopomers of side-chain amides are as significant as on backbone amides. (c) 2007 Elsevier lnc. All rights reserved.