Precise vicinal coupling constants³J_HNα in proteins from nonlinear fits of J-modulated [¹⁵N,¹H]-COSY experiments

Improved experimental schemes for the recently introduced J-modulated [¹⁵N,¹H]-correlation experiment for measurements of the homonuclear amide proton-C^α proton vicinal coupling constants.³J_HNα, in uniformly¹⁵N-labeled proteins are described, and a nonlinear fit procedure is presented for quantitative evaluation of³J_HNα. The method was first tested with the N-terminal DNA-binding domain of the 434 repressor (M=7.3 kDa), where at 13 C precise values of³J_HNα in the range 2.0-9.5 Hz were obtained for all residues with resolved¹⁵N-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 ∼ 18 kDa). The³J_HNα values measured were found to be in excellent agreement with those predicted from the secondary structure of this protein in the complex.