Multiple-Quantum Line Narrowing for Measurement of H^α—H^βJ Couplings in Isotopically Enriched Proteins

Uniform ¹³C enrichment of proteins is commonly used for NMR studies of proteins that are not amenable to conventional homonuclear 2D NMR spectroscopy. In such studies, the one-bond ¹H—¹³C dipolar interaction is usually the dominant source of ¹H line broadening. ¹H—¹³C zero- and double-quantum coherences are, to first order, not affected by this dipolar relaxation mechanism. The relatively long relaxation time of such ¹H^α—¹³C^αmultiple-quantum coherences is exploited for measurement of H^α—H^βJ couplings in a sample of uniformly ¹³C-enriched calcium-free calmodulin (16.7 kD) and a sample of TGF-β1 (25 kDa). 7(H^α—H^β) provides information on the stereospecific resonance assignment for residues with nonequivalent H^βmethylene protons and on the χ₁ torsion angles.