Methods for the measurement of 1JNCα and 2JNCα from a simplified 2D 13Cα-coupled 15N SE-HSQC spectrum

Two methods for the measurement of (2)J(NC alpha) and (1)J(NC alpha) in N-15/ C-13-labeled small and medium-size proteins are described. The current approach is based on simplified C-13(alpha)-coupled N-15 HSQC spectra, where the two (2)J(NC alpha) doublets are separated into two subspectra corresponding to the alpha and beta spin states of the residue's own alpha carbon. The displacement of the two (2)J(NC alpha) doublets between the two subspectra provides an accurate value for (1)J(NC alpha). The alpha/beta filtration is achieved by taking the sum and difference of the recorded complementary in-phase and antiphase J-coupled spectra. J-multiplication is utilized in one of the proposed methods. In this method, an additional coupling evolution period, which is incremented in concert with t(1), is included in the pulse sequence making it possible to scale the peak-to-peak separation. (C) 2001 Academic Press.