A PRACTICAL METHOD FOR STEREOSPECIFIC ASSIGNMENTS OF GAMMA-METHYLENE AND DELTA-METHYLENE HYDROGENS VIA ESTIMATION OF VICINAL H-1-H-1 COUPLING-CONSTANTS

Stereospecific assignments are made for gamma- and delta-methylene hyd rogens in a protein by means of estimation of vicinal H-1-H-1 spin-spi n coupling constants from a short-mixing-time TOCSY experiment. (3)J(a lpha beta) coupling constants, as measured from a P.E. COSY map, are s hown to be well correlated with alpha-beta cross-peak intensities of a short-mixing-time (10 ms) TOCSY map. The procedure is illustrated by application to a trypsin-inhibitor protein (M(r) similar to 7 Kd). Thu s, gamma-methylene hydrogens of isoleucine residues have been stereosp ecifically assigned on the basis of (3)J(beta gamma) H-1-H-1 coupling patterns and intraresidue cross-peak intensities in a NOESY map; gamma -hydrogens of other residues, such as lysine and arginine, have been s tereospecifically assigned solely on the basis of cross-peak intensity patterns resulting from coupling of two beta-hydrogens to two gamma-h ydrogens, and in conjunction with stereospecific assignments of beta-m ethylene hydrogens. However, intraresidue NOE intensities are needed i f one or two pairs of coupling constants cannot be estimated because o f cross peaks either overlapping or occurring proximal to the diagonal . The delta-methylene hydrogens have been stereospecifically assigned on the basis of coupling between two gamma-hydrogens and two delta-hyd rogens, in combination with stereospecific assignments of gamma-hydrog ens. Stereospecific assignments of side chains should contribute to th e overall precision and accuracy of NMR-determined three-dimensional s olution structures of proteins. (C) 1995 Academic Press, Inc.