APPLICATION OF H(N)CA,CO-E.COSY EXPERIMENTS FOR CALIBRATING THE PHI-ANGULAR DEPENDENCES OF VICINAL COUPLINGS J(C'(I-1),H-ALPHA(I)), J(C'(I-1),C-I(BETA)) AND J(C'(I-1),C'(I)) IN PROTEINS

A triple-resonance NMR technique suitable for the determination of car bonyl-related couplings in polypeptide systems is introduced. The appl ication of three novel pulse sequences to uniformly C-13/N-15-enriched proteins yields E.COSY-like multiplet patterns exhibiting either one of the (3)J(Ci-l',H-i(alpha)), (3)J(Ci-l',C-i(beta)) and (3)J(Ci-l',C- i') coupling constants in the indirectly detected C-13' dimension, dep ending on the passive spin selected. The experiments are demonstrated with oxidized flavodoxin from Desulfovibrio vulgaris On the basis of t he J-values measured and the backbone phi-angles derived from a high-r esolution X-ray structure of the protein, the three associated Karplus equations were reparametrized. The root-mean-square differences betwe en the experimental coupling constants and those predicted by the opti mized Karplus curves are 0.41, 0.33 and 0.32 Hz for (3)J(Ci-l',H-i(alp ha)), (3)J(Ci-l',C-i(beta)) and (3)J(Ci-l',C-i'), respectively The res ults are compared with the Karplus parameters previously published for the same couplings.