Structural Characterization of Metallothionein by Multinuclear NMR

Abstract

The application of ¹H, ¹³C, and ¹¹³Cd Fourier transform NMR to the study of Cd, Zn metallothionein offers the promise of providing the detailed structural information concerning the chemical nature of the metal binding sites which has heretofore been lacking. Among the several factors which make the study of this protein by multinuclear NMR particularly attractive are its ready availability in relatively large amounts from animals injected with cadmium and its small molecular weight, which should allow reasonably well-resolved high field natural abundance ¹³C and ¹H spectra to be obtained. ¹H NMR spectra of metallothionein at 270 MHz have been reported and show several spectral changes to occur in the aliphatic region induced by metal ion removal at low pH (1). Unfortunately, assignment of these spectral perturbations to structural events occurring at specific sites in the protein is difficult due to the inability to resolve individual resonances from the large number of overlapping signals confined to the relatively narrow ¹H chemical shift range. The prospects for resolution and assignment of individual ¹³C resonances are much better, however, due to the wide chemical shift range of the ¹³C nucleus and the absence of spin-spin splitting when H decoupling is employed. Recent work at high magnetic field strengths has demonstrated that even in the aliphatic region, numerous resolved single carbon resonances are obtained in ¹³C NMR spectra of proteins larger than metallothionein (2,3).