The use of 1JC?H? coupling constants as a probe for protein backbone conformation

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Abstract
Summary Simple pseudo-3D modifications to the constant-time HSQC and HCACO experiments are described that allow accurate (±0.5 Hz) measurement of one bond JCαHα coupling constants in proteins that are uniformly enriched with 13C. An empirical φ,ψ-surface is calculated which describes the deviation of 1JCαHα from its random coil value, using 203 1JCαHα values measured for residues in the proteins calmodulin, staphylococcal nuclease, and basic pancreatic trypsin inhibitor, for which φ and ψ are know with good precision from previous X-ray crystallographic studies. Residues in α-helical conformation exhibit positive deviations of 4–5 Hz, whereas deviations in β-sheet are small and, on average, slightly negative. Data indicate that 1JCαHα depends primarily on ψ, and that 1JCαHα may be useful as a qualitative probe for secondary structure. Comparison of 1JCαHα coupling constants measured in free calmodulin and in its complex with a 26-aminoacid peptide fragment of myosin light-chain kinase confirm that the calmodulin secondary structure is retained upon complexation but that disruption of the middle part of the ‘central helix’ is even more extensive than in free calmodulin.

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