PRECISE VICINAL COUPLING-CONSTANTS 3JHN-ALPHA IN PROTEINS FROM NONLINEAR FITS OF J-MODULATED [N-15,H-1]-COSY EXPERIMENTS

Improved experimental schemes for the recently introduced J-modulated [N-15, H-1]-correlation experiment for measurements of the homonuclear amide proton-C(alpha) proton vicinal coupling constants, 3J(HN-alpha), in uniformly N-15-labeled proteins are described, and a nonlinear fit procedure is presented for quantitative evaluation of 3J(HN-alpha). The method was first tested with the N-terminal DNA-binding domain of the 434 repressor (M = 7.3 k Da), where at 13-degrees-C precise values of 3J(HN-alpha) in the range 2.0-9.5 Hz were obtained for all residues with resolved N-15-H-1 cross peaks. It was then applied to the Antennapedia homeodomain complexed to a synthetic 14-base pair DNA fragment (molecular weight of the complex approximately 18 kDa). The 3J(HN-alpha) values measured were found to be in excellent agreement with those predicted from the secondary structure of this protein in the complex.