Systematic application of high-resolution, phase-sensitive two-dimensional 1H-NMR techniques for the identification of the amino-acid-proton spin systems in proteins. Rabbit metallothionein-2.

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.