Résumé
A new and convenient method, named ARTSY-j, is introduced that permits extraction of the (3)J(HNH alpha) couplings in proteins from the relative intensities in a pair of N-15-H-1 TROSY-HSQC spectra. The pulse scheme includes (3)J(HNH alpha) dephasing of the narrower TROSY H-1(N)-{N-15) doublet component during a delay, integrated into the regular two-dimensional TROSY-HSQC pulse scheme, and compares the obtained intensity with a reference spectrum where (3)J(HNH alpha) dephasing is suppressed. The effect of passive H-1(alpha) spin flips downscales the apparent (3)J(HNH alpha) coupling by a uniform factor that depends approximately linea(r)ly on both the duration of the (3)J(HNH alpha) dephasing delay and the H-1-H-1 cross relaxation rate. Using such a correction factor, which accounts for the effects of both inhomogeneity of the radiofrequency field and H-1(alpha) spin flips, agreement between prior and newly measured values for the small model protein GB3 is better than 0.3 Hz. Measurement for the HIV-1 protease homodimer (22 kDa) yields (3)J(HNH alpha) values that agree to better than 0.7 Hz with predictions made on the basis of a previously parameterized Karplus equation. Although for Gly residues the two individual (3)J(HNH alpha) couplings cannot be extracted from a single set of ARTSY-J spectra, the measurement provides valuable phi angle information. Published by Elsevier Inc.