Application of HN(C)N to rapid estimation of ¹J(N-C^α) coupling constants correlated to ψ torsion angles in proteins: implication to structural genomics

Abstract

We recently described a triple resonance experiment, HN(C)N, for sequential correlation of H^N and ¹⁵N atoms in (¹⁵N, ¹³C) labeled proteins [J. Biomol. NMR. 20 (2001) 135]. Here, we describe an approach based on this experiment for estimation of one bond N-C^α J-couplings in medium size labeled proteins, which seem to show good correlations with ψ torsion angles along the protein backbone. The approach uses the ratio of the intensities of the sequential and diagonal peaks in the F₂-F₃ planes of the HN(C)N spectrum. The reliability of the approach has been demonstrated using a short peptide wherein the coupling constants have been measured by the present method and also independently from peak splittings in HSQC spectra. The two results agree within 10%. The applicability of the procedure to proteins has been demonstrated using doubly labeled FK506 binding protein (FKBP, molecular mass ~12 kDa). Coupling constant estimates have been obtained for 62 out of 100 non-proline residues and they show a correlation with ψ torsion angles, as has been reported before. This semi-quantitative application of HN(C)N extends the significance of the experiment especially, in the context of structural genomics, since the single experiment, not only provides a great enhancement in the speed of resonance assignment, but also provides quantitative structural information.