DETERMINATION OF BACKBONE NITROGEN-NITROGEN J-CORRELATION IN PROTEINS

Recently, a quantitative J correlation technique has been presented wh ich makes use of homonuclear Hartmann-Hahn cross-polarization (TOCSY) to measure (3)J(C'C') in proteins isotopically enriched with C-13 [Grz esiek, S. and Bax, A. (1997) J. Biomol. NMR, 9, 207-211]. Since homonu clear Hartmann-Hahn is twice as fast as conventional COSY transfer, th is method is much less sensitive to transverse relaxation, which is th e principal limiting factor in achieving long-range J-coupling correla tions in macromolecules. Here we describe a similar experiment which i s used to measure (3)J(NN) coupling constants between sequential amide N-15 nuclei in the backbone of ubiquitin. As expected from the low ma gnetic moment of N-15, the (3)J(NN) coupling constants are exceedingly small, with values between 0.14 and 0.36 Hz for residues in beta-conf ormations and values below 0.15 Hz for residues in alpha-conformations . In contrast to what is expected from a Karplus-type dependence on th e backbone angle psi, large differences in the values of (3)J(NN) are observed for a number of residues with very similar backbone psi angle s. A quantitative description of statistical and systematic errors, in particular of relaxation effects during the TOCSY transfer, shows tha t these differences are highly significant.