A 2-DIMENSIONAL NMR-STUDY OF CO(II)(7) RABBIT LIVER METALLOTHIONEIN

The 600-MHz H-1-NMR NOESY spectra on Co(II)7-reconstituted metallothionein (Co7MT), exhibiting hyperfine signals in the range 350 ppm to - 50 ppm, with nuclear relaxation times of the order of a few milliseconds, have been measured and several interproton connectivities have been detected. To our knowledge, this is the largest spectral window ever reported for a two-dimensional H-1-NMR spectrum in the case of a paramagnetic metalloprotein. No scalar connectivities could be detected. The hyperfine-shifted signals belong to the cysteine-ligand protons of the Co4S11 cluster Of Co7MT. Together with results from one-dimensional NOE experiments, the two-dimensional experiments allowed us to proceed with the pairwise assignment of the isotropically shifted signals of the (CbetaH)H-2 groups of the metal-coordinated cysteines. With the aid of computer-graphics inspection of the four-metal-cluster domain, based on the NMR solution structure of Cd7MT, it is possible to propose sequence-specific assignments of a few hyperfine-shifted H-1-NMR signals. In particular, a tentative assignment is given for the six signals whose shifts exhibit an antiCurie temperature dependence. The assignment relies on the theoretical model that qualitatively rationalizes the isotropic-shift pattern and its temperature dependence. Inferences on the solution structure of the Co4S11 cluster are drawn.