Determination of backbone nitrogen–nitrogen J correlations in proteins

Abstract

Recently, a quantitative J correlation technique has been presented which makes use of homonuclear Hartmann–Hahn cross-polarization (TOCSY) to measure ³J_C′_C′ in proteins isotopically enriched with ¹³C [Grzesiek, S. and Bax, A. (1997) J. Biomol. NMR, 9, 207–211]. Since homonuclear Hartmann–Hahn is twice as fast as conventional COSY transfer, this method is much less sensitive to transverse relaxation, which is the principal limiting factor in achieving long-range J-coupling correlations in macromolecules. Here we describe a similar experiment which is used to measure³ J_NN coupling constants between sequential amide¹⁵ N nuclei in the backbone of ubiquitin. As expected from the low magnetic moment of ¹⁵N, the ³J_NN coupling constants are exceedingly small, with values between 0.14 and 0.36 Hz for residues in β-conformations and values below 0.15 Hz for residues in α-conformations. In contrast to what is expected from a Karplus-type dependence on the backbone angle ψ, large differences in the values of³ J_NN are observed for a number of residues with very similar backbone ψ angles. A quantitative description of statistical and systematic errors, in particular of relaxation effects during the TOCSY transfer, shows that these differences are highly significant.