Application of H(N)CA,CO-E.COSY experiments for calibrating the φ angular dependences of vicinal couplings J(C′_i−1,H_i ^α), J(C′_i−1,C_i ^β) and J(C′_i−1,C′_i) in proteins

Abstract

A triple-resonance NMR technique suitable for the determination ofcarbonyl-related couplings in polypeptide systems is introduced. Theapplication of three novel pulse sequences to uniformly¹³C/¹⁵N-enriched proteins yields E.COSY-likemultiplet patterns exhibiting either one of the³J(C′_i−1,H_i ^α), ³J(C′_i−1,C_i ^β) and³J(C′_i−1,C′_i)coupling constants in the indirectly detected ¹³C′dimension, depending on the passive spin selected. The experiments aredemonstrated with oxidized flavodoxin from Desulfovibrio vulgaris. On thebasis of the J-values measured and the backbone φ-angles derived from ahigh-resolution X-ray structure of the protein, the three associated Karplusequations were reparametrized. The root-mean-square differences between theexperimental coupling constants and those predicted by the optimized Karpluscurves are 0.41, 0.33 and 0.32 Hz for³J(C′_i−1,H_i ^α),³J(C′_i−1,C_i ^β) and³J(C′_i−1,C′_i),respectively. The results are compared with the Karplus parameters previouslypublished for the same couplings.