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