CONFORMATION OF VALINE SIDE-CHAINS IN RIBONUCLEASE T-1 DETERMINED BY NMR-STUDIES OF HOMONUCLEAR AND HETERONUCLEAR (3)J COUPLING-CONSTANTS

A conformational analysis of the valine side chains of ribonuclease T-1 (RNase T-1) was performed using NMR spectroscopy, in particular homonuclear (H-1,H-1 and C-13,C-13) and heteronuclear (H-1,N-15 and H-1,C-13) vicinal spin-spin coupling constants as obtained from E.COSY-type NMR experiments. The coupling constants related to the (chi 1) dihedral angle in valine, (3)J(H alpha H beta),(3)J(NH beta),(3)J(C'H beta),(3)J(H alpha C gamma 1),(3)J(H alpha C gamma 2),(3)J(C'C gamma 1) and (3)J(C'C gamma 2), were evaluated in a quantitative manner. The analysis of (3)J data allowed for the stereospecific assignment of the valine methyl resonances. On the basis of various models for motional averaging of coupling constants, a fit of the torsion angles (chi 1) to a set of the experimental (3)J coupling constants ((3)J(H alpha)H(beta), (3)J(NH beta), (3)J(C'H beta)) was carried out. The resulting side-chain conformations were examined with respect to NOE distance informations. Single rotameric states emerged for Val16, Val67, Val79, and Val101, while conformational equilibria between staggered rotamers were found for Val33 and Val78. Using a different model approach, Val52 and Val89 are also likely to exhibit unimodal (chi 1) angle distributions. The analysis was found to depend critically on the set of Karplus parameters used. Except for Val52 and Val78, the predominant rotamers obtained from (3)J coupling informations agree with the conformation in the crystal structure of ribonuclease T-1 (Martinez-Oyanedel et al., 1991).