MAGNETIC-FIELD DEPENDENCE OF NITROGEN-PROTON J-SPLITTING IN N-15-ENRICHED HUMAN UBIQUITIN RESULTING FROM RELAXATION INTERFERENCE AND RESIDUAL DIPOLAR COUPLING

One-bond (1)J(NH) couplings have been measured in N-15-enriched human ubiquitin and range from 91.1 to 95.6 Hz. Measurements have been carri ed out using two different methods and at H-1 frequencies of 360, 500, and 600 MHz. The best method yields a precision of ca 0.02 Hz, and pe rmits reliable measurement of the small changes (<0.3 Hz) in (1)J(NH) Splitting that occur when the magnetic field strength is increased fro m 8.5 to 14 T. The dependence of the (1)J(NH) splittings on the streng th of the static magnetic field originates from two sources: a dynamic frequency shift caused by interference of the N-15 chemical shift ani sotropy and the N-15-H-1 dipolar coupling relaxation mechanisms, and a dipolar contribution caused by a small degree of alignment resulting from the anisotropic magnetic susceptibility of the diamagnetic protei n. Best fitting of the measured data yields an orientation-independent decrease of 0.11 Hz in the (1)J(NH) splittings at 609 MHz relative to 360 MHZ; in perfect agreement with theoretical predictions for the ma gnitude of the dynamic frequency shift. When fitting the measured J va lues to the theoretical model, containing only the dynamic frequency s hift and dipolar coupling contributions, the reduced error in the stat istical F-test is smaller than one, assuming a 0.02 Hz rms error in th e experimental (1)J(NH) splittings. This confirms that the random erro r in the measured data J(NH) values does not exceed 0.02 Hz, and that effects other than the dipolar coupling and dynamic frequency shift ar e not detectable. Dependence of the change in (1)J(NH) On the orientat ion of the N-H bond vector within the molecular frame yields experimen tally determined axial and rhombic magnetic shielding susceptibility a nisotropies of -2.1 x 10(-28) and 0.7 x 10(-28) cm(3)/molecule, respec tively. A small improvement of the fit is observed when the amide prot on is positioned at a distance above or below the C-i-1'-N-i-C-i(alpha ) plane which is about five times smaller than the out-of-plane distan ce predicted by ab initio calculations on a dipeptide analog in vacuum .