Methods for the measurement of (1)J(NC alpha) and (2)J(NC alpha) from a simplified 2D C-13(alpha)-coupled N-15 SE-HSQC spectrum

Two methods for the measurement of (2)J(NC alpha) and (1)J(NC alpha) in N-1 5/ C-13-labeled small and medium-size proteins are described. The current a pproach 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 correspo nding 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 subspec tra provides an accurate value for (1)J(NC alpha). The alpha/beta filtratio n is achieved by taking the sum and difference of the recorded complementar y in-phase and antiphase J-coupled spectra. J-multiplication is utilized in one of the proposed methods. In this method, an additional coupling evolut ion period, which is incremented in concert with t(1), is included in the p ulse sequence making it possible to scale the peak-to-peak separation. (C) 2001 Academic Press.