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It is well known that the magnitude of heteronuclear long-range couplings contains useful information regarding molecular conformation (2). For measurement of such couplings between protons and protonated heteronuclei, E. COSY-based techniques (2) in principle can yield quantitative results in isotopically enriched proteins (3-6). For measurement of J couplings to nonprotonated heteronuclei, deconvolution of the cross-peak shape in a ‘H-detected heteronuclear multiple-bond correlation (HMBC) spectrum (7) can be used (8-10)) although for larger proteins the ‘H linewidth and extensive spectral overlap render this approach difficult, if not impossible. Alternatively, qualitative information about the size of a long-range coupling can be obtained from the intensity of correlations in a 3D spectrum in which magnetization is transferred via the long-range coupling of interest. This has recently been demonstrated for couplings between 15N and intraresidue HP protons (11, 12). Here we present an analogous experiment that uses magnetization transfer via the J coupling between the carbonyl (CO) and the H/3 protons. The resulting information about the CO-HP coupling, in combination with knowledge of the HP-15N J coupling and qualitative (13) or quantitative (2) data on the Ha-HB couplings, allows unambiguous stereospecific assignment of the HP methylene protons and determines the rotomeric state of the CWCP bond (xi angle).

The pulse sequence of our new experiment, which relies on magnetization transfer via the multiple-bond ‘H-i3C0 J coupling, is sketched in Fig. 1. Because its main function is to correlate amide protons and 15N nuclei …