Systematic application of high‐resolution, phase‐sensitive two‐dimensional 1H‐NMR techniques for the identification of the amino‐acid‐proton spin systems in proteins

Abstract

Novel strategies for elucidation and classification of amino acid ¹H-NMR spin systems in proteins were developed exploiting recently introduced two-dimensional NMR techniques such as phase-sensitive double-quantum-filtered correlated spectroscopy, relayed coherence transfer spectroscopy, double quantum spectroscopy and nuclear Overhauser spectroscopy. Due to the improved resolution in phase-sensitive spectra, the fine structure of cross peaks could be exploited as a powerful source of information for establishing ¹H-¹H connectivities. Principles for the interpretation of multiplet structures of absorption mode cross peaks are discussed. With these methods the ¹H spin systems of rabbit liver metallothionein-2 were elucidated and classified according to amino acid types. Despite the intrinsically difficult situation arising from the unusual amino acid composition of this protein, a more complete characterization of the ¹H spin systems prior to the step of sequential resonance assignments was achieved with the presently introduced methodology than was possible in earlier studies of proteins of similar size.