Improved measurement of (3)J(H-i(alpha), Ni+1) coupling constants in H2O dissolved proteins

A modification to the recently proposed alpha/beta -HN(CO)CA-J TROSY pulse sequence (P. Permi et al, J. Magn. Reson. 146, 255-259 (2000)) makes it pos sible to determine (3)J(H-i(alpha), Ni+1) coupling constants from a single E.COSY-type cross-peak pattern rather than from two H-1(alpha) spin-state-e dited subspectra. Advantages are increased N-15 resolution, critical to ext racting accurate H-1(alpha)-N-15 coupling constants, and minimized differen tial relaxation due to nested C-13(alpha) and N-15 evolution periods. Appli cation of the improved pulse sequence to Desulfovibrio vulgaris flavodoxin results in (3)J(H-i(alpha), Ni+1) values being systematically larger than t hose obtained with the original scheme. Parametrization of the coupling dep endence on the protein backbone torsion angle psi yields the Karplus relati on (3)J(H-i(alpha), Ni+1) = -1.00 cos(2)(psi - 120 degrees) + 0.65 cos(psi - 120 degrees)-0.15 Hz, with a residual root-mean-square difference of 0.13 Hz between measured and back-calculated coupling constants. The curve comp ares with data derived from ubiquitin (A. C. Wang and A. Bax, J. Am. Chem. Soc. 117, 1810-1813 (1995)), although spanning a slightly larger range of J values in flavodoxin. The orientation of the Ala39/Ser40 peptide link, for ming a type-II beta -turn in flavodoxin, is twisted against X-ray-derived t orsions by approximately 10 degrees in the NMR structure as evident from th e analysis of phi- and psi -related (3)J coupling constants. The remaining deviation of some experimental values from the prediction is likely to be d ue to strong hydrogen bonding, substituent effects, or the additional depen dence on the adjacent torsions phi. (C) 2001 Academic Press.