Simple Approaches for Estimating Vicinal 1H- 1H Coupling-Constants and for Obtaining Stereospecific Resonance Assignments in Leucine Side Chains

Abstract

An approach for deriving stereospecific δ-methyl assignments and χ 2 dihedral angle constraints for leucine residues, based on easily recognized patterns of 1H- 1H spin-spin coupling constants and intraresidue nuclear-Overhauser-effect spectroscopy (NOESY) cross-peak intensities, is described. The approach depends on resolved H γ and/or δ-methyl resonances and on initially obtaining stereospecific assignments for H β2 and H β3. As part of the overall strategy, a method is presented for obtaining qualitative or, in favorable cases, semiquantitative estimates of vicinal 1H- 1H coupling constants from peak intensities measured in a short-mixing-time 1H- 1H total correlation spectroscopy (TOCSY) experiment. This method of estimating 1H- 1H spin-spin coupling constants is generally applicable to all side-chain types. The approach is illustrated for several leucine residues within uniformly 15N-labeled and 15N/ 13C-double-labeled isolated light-chain variable domain of the anti-digoxin antibody 26-10. Estimates of 3 J αβ and 3 J βγ coupling constants are derived from a three-dimensional (3D) 13C-edited TOCSY-heteronuclear multiple-quantum coherence (HMQC) spectrum. These data are combined with information from 3D 15N-edited NOESY and 3D 13C-edited NOESY spectra to yield stereospecific H β2, H β3, and δ-methyl assignments, as well as constraints on χ (1) and χ 2 dihedral angles. Although the overall approach is illustrated using 3D 15N-edited and 13C-edited data, it is equally applicable to analysis of two-dimensional 1H- 1H NOESY and TOCSY spectra.