A two-dimensional NMR study of Co(II)7 rabbit liver metallothionein

Eur J Biochem. 1993 Jan 15;211(1-2):235-40. doi: 10.1111/j.1432-1033.1993.tb19891.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cations, Divalent
  • Cobalt
  • Cysteine / chemistry
  • In Vitro Techniques
  • Liver / chemistry
  • Magnetic Resonance Spectroscopy
  • Metallothionein / chemistry
  • Metallothionein / ultrastructure*
  • Protein Conformation
  • Rabbits

Substances

  • Cations, Divalent
  • Cobalt
  • Metallothionein
  • Cysteine