SIMPLE APPROACHES FOR ESTIMATING VICINAL H-1-H-1 COUPLING-CONSTANTS AND FOR OBTAINING STEREOSPECIFIC RESONANCE ASSIGNMENTS IN LEUCINE SIDE-CHAINS

An approach for deriving stereospecific delta-methyl assignments and c hi(2) dihedral angle constraints for leucine residues, based on easily recognized patterns of H-1-H-1 spin-spin coupling constants and intra residue nuclear-Overhauser-effect spectroscopy (NOESY) cross-peak inte nsities, is described. The approach depends on resolved H-gamma and/or delta-methyl resonances and on initially obtaining stereospecific ass ignments for H-beta 2 and H-beta 3. As part of the overall strategy, a method is presented for obtaining qualitative or, in favorable cases, semiquantitative estimates of vicinal H-1-H-1 coupling constants from peak intensities measured in a short-mixing-time H-1-H-1 total correl ation spectroscopy (TOCSY) experiment. This method of estimating H-1-H -1 spin-spin coupling constants is generally applicable to all side-ch ain types. The approach is illustrated for several leucine residues wi thin uniformly N-15-labeled and N-15/C-13-double-labeled isolated ligh t-chain variable domain of the anti-digoxin antibody 26-10. Estimates of (3)J(alpha beta) and (3)J(beta gamma) coupling constants are derive d from a three-dimensional (3D) C-13-edited TOCSY-heteronuclear multip le-quantum coherence (HMQC) spectrum. These data are combined with inf ormation from 3D N-15-edited NOESY and 3D C-13-edited NOESY spectra to yield stereospecific H-beta 2, H-beta 3, and delta-methyl assignments , as well as constraints on chi(1) and chi(2) dihedral angles. Althoug h the overall approach is illustrated using 3D N-15-edited and C-13-ed ited data, it is equally applicable to analysis of two-dimensional H-1 -H-1 NOESY and TOCSY spectra. (C) 1994 Academic Press, Inc.