A general approach for determining scalar coupling constants in polypeptides and proteins

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Abstract
A general approach is described for measuring homo‐ and heteronuclear spin coupling constants in polypeptides and small proteins. This method uses selective magnetization transfer to generate cross peaks similar to exclusive correlated spectroscopy (E.COSY) and a large direct spin coupling (1J) in one dimension to “pull apart” cross‐peak components by frequencies much larger than the resonance line width. A general description of this method is presented, along with a brief discussion of spin topology and relaxation effects that must be considered in designing multidimensional nmr pulse sequences for measuring vicinal coupling constants. The principles are demonstrated in designs of several two‐dimensional nmr experiments for determining coupling constants in polypeptides and proteins. These include experiments for measuring 3J(HN‐Hα), 3J(H ‐15Ni), 3J(15N‐Hβ), and 3J(Hα‐Hβ) coupling constants, which depend on the polypeptide dihedral angles ϕ, ψ and χ1. Multidimensional nmr experiments developed with this approach will allow measurements of many vicinal coupling constants in peptides, proteins, and other molecules. Coupling constants measured in these spectra can be used to determine backbone and side‐chain conformations, to obtain stereospecific resonance assignments of prochiral atoms, and to characterize conformational distributions of dihedral angles. Combined with information obtained from nuclear Overhauser effect measurements, these data will provide more precise determinations of protein solution structures by nmr spectroscopy.