A Practical Method for Stereospecific Assignments of γ- and δ-Methylene Hydrogens via Estimation of Vicinal 1H-1H Coupling Constants

Stereospecific assignments are made for γ- and δ-methylene hydrogens in a protein by means of estimation of vicinal 1H-1H spin-spin coupling constants from a short-mixing-time TOCSY experiment. 3Jαβ coupling constants, as measured from a P.E. COSY map, are shown to be well correlated with α-β cross-peak intensities of a short-mixing-time (10 ms) TOCSY map. The procedure is illustrated by application to a trypsin-inhibitor protein (Mr 7 Kd). Thus, γ-methylene hydrogens of isoleucine residues have been stereospecifically assigned on the basis of 3Jβγ1H-1H coupling patterns and intraresidue cross-peak intensities in a NOESY map; γ-hydrogens of other residues, such as lysine and arginine, have been stereospecifically assigned solely on the basis of cross-peak intensity patterns resulting from coupling of two β-hydrogens to two γ-hydrogens, and in conjunction with stereospecific assignments of β-methylene hydrogens. However, intraresidue NOE intensities are needed if one or two pairs of coupling constants cannot be estimated because of cross peaks either overlapping or occurring proximal to the diagonal. The δ-methylene hydrogens have been stereospecifically assigned on the basis of coupling between two γ-hydrogens and two δ-hydrogens, in combination with stereospecific assignments of γ-hydrogens. Stereospecific assignments of side chains should contribute to the overall precision and accuracy of NMR-determined three-dimensional solution structures of proteins.