A two-dimensional NMR study of Co(II)7 rabbit liver metallothionein.
The 600-MHz 1H-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 1H-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 C beta H2 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 purpose sequence-specific assignments of a few hyperfine-shifted 1H-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.
Bertini
I
I
Department of Chemistry, University of Florence, Italy.
Luchinat
C
C
Messori
L
L
Vasak
M
M
D000818:Animals; D002413:Cations, Divalent; D003035:Cobalt; D003545:Cysteine / Q000737:chemistry; D066298:In Vitro Techniques; D008099:Liver / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008668:Metallothionein / Q000737:chemistry / Q000648:ultrastructure*; D011487:Protein Conformation; D011817:Rabbits
D002413:Cations, Divalent; D003035:Cobalt; D008668:Metallothionein; D003545:Cysteine