Cadmium-113 nuclear magnetic resonance ("13Cd NMR) has been used to determine the structures of the multiple cadmium binding sites in the two major isoproteins of rabbit liver metallothionein. The isotopically "13Cd-labeled metallothionein used in these studies was isolated from the livers of rabbits that had been subjected to repeated injections of "13CdCl2. The native protein isolated from these livers contains an appreciable amount of Zn in addition to Cd, ranging from 2-3 mol per mol of protein out of a total metal content of 7 mol per mol of protein. The "13Cd NMR spectrum of Cd, Zncontaining metallothionein is quite complex, reflecting the fact that the native protein is a heterogeneous mixture of species containing different relative amounts of Zn and Cd. Replacement of the native Zn with "13Cd in vitro gave a protein whose "13Cd NMR spectrum was much simpler, containing eight distinct multiplets with chemical shifts ranging from 611-670 ppm. The origin of the multiplet structures has been shown to be "13Cd-''3Cd scalar cou Fing arising from two-bond interactions between "13Cd ions linked to one another by bridging cysteine thiolate ligands. The size and structures of the metal clusters in the protein were determined by the application of selective homonuclear "13Cd decou ling techniques. Analysis of these data showed that rabbit iver metallothionein contains two separate metal clusters, one containing four Cd2+ ions and the other containing three. These two clusters, whose structures are the same in both isoproteins, have been designated "cluster A" and "cluster B," respectively. Structures for the clusters are proposed that account for the "13Cd spin coupling data and the participation of all 20 of the cysteine residues in metal ligation, 11 in cluster A and 9 in cluster B. The appearance in the spectrum of eight multiplets rather than the seven that would be expected on the basis of the number of metal binding sites in the protein is an indication of some residual heterogeneity in the 113Cd-labeled metallothionein sample. The origin of this heterogeneity is suggested to be the presence of a protein species that lacks metal ions at its cluster B binding sites.Metallothioneins are a ubiquitous class of sulfhydryl-rich, low molecular weight proteins that are believed to play a central role in metal metabolism. Since the initial discovery in 1957 of the cadmium-and zinc-containing proteins from equine kidney cortex (1), metallothioneins have been characterized from a variety of other mammals (including man) and from such widely divergent sources as birds, fish, microorganisms, and invertebrates (2, 3). The widespread occurrence of metallothionein in nature suggests that it serves an important biological function, though its exact role has yet to be defined. Most attention has centered on the hypothesis that the protein acts as a heavy metal detoxifying agent by sequestering cadmium, mercury, and other harmful metal ions. Bolstering this suggestion is the observation that exposure of many organisms ...

Metallothioneins (MTs) and various other metal binding proteins release metals when exposed to nitric oxide (NO). We investigated the structural consequences of the interaction between MTs and NO by using 1H‐ and 113Cd‐NMR spectroscopy and found that only the three metals from the N‐terminal β‐domain were selectively released whereas the C‐terminal α‐domain remains intact. Since it has been proposed that the β‐domain is responsible for the postulated role of MTs in zinc homeostasis, whereas the tight binding of metals in the α‐domain appears to play a role in heavy metal detoxification, our results suggest a potential regulatory role of NO in zinc distribution. Specifically, we present a mechanism whereby MT counteracts the cytotoxic effects of NO at inflammatory sites.

Metallothionein is a cysteine-rich metal-binding protein whose biosynthesis is closely regulated by the level of exposure of an organism to zinc, copper, cadmium, and other metal salts. The metallothionein from Callinectes sapidus is known to bind six divalent metal ions in two separate metal-binding clusters. Heteronuclear 1H-113Cd and homonuclear 1H-1H NMR correlation experiments have been used to establish that the two clusters reside in two distinct protein domains. The three-dimensional solution structure of the metallothionein has been determined using the distance and angle constraints derived from these two-dimensional NMR data sets and a distance geometry/simulated annealing protocol. There are no interdomain short distance (< or = 4.5 A) constraints observed in this protein, enabling the calculation of structures for the N-terminal, beta domain and the C-terminal, alpha domain separately. A total of 18 structures were obtained for each domain. The structures are based on a total of 364 experimental NMR restraints consisting of 277 approximate interproton distance restraints, 12 chi 1 and 51 phi angular restraints, and 24 metal-to-cysteine connectivities obtained from 1H-113Cd correlation experiments. The only element of regular secondary structure in either of the two domains is a short segment of helix in the C-terminal alpha domain between Lys42 and Thr48. The folding of the polypeptide backbone chain in each domain, however, gives rise to several type I beta turns. There are no type II beta turns.

3D solution structural calculations for yeast silver(1)-substituted metallothionein (MT) and native copper(I) MT were completed using experimentally determined NOE and dihedral angle constraints, in conjunction with experimentally derived metal-to-Cys connectivities for AgMT which were assumed identical for CuMT. For the first 40 residues in both structures, the polypeptide backbone wraps around the metal cluster in two large parallel loops separated by a deep cleft containing the metal cluster. Minor differences between the two structures include differences in hydrogen bonds and the orientation of the N-terminus with the overall protein volume conserved to within 6.5%.

9354laxation dela s, and filtered identically, depends on the ratio of the two bandwidthsd If the audio filter cutoffs are set at the Nyquist frequency, that ratio becomes the ratio of the acquisition times (fzmu). The relative sensitivity is approximately proportional to the ratio of the acquisition times divided by the ratio of the delays between pulses.20 When the sweep width is reduced while acquiring the same number of sample points, the acquisition time increases from 102.4 to 256 ms; however, the delay between pulses, -2.5 s, does not change significantly. Consequently, the sensitivity is improved by the square root of the ratio of acquisition times, (256/ I02.4)l/~ -60%. Intuitively, the signaI/noise ratio improves because the noise bandwidth is reduced; the sensitivity increases because a larger portion of the spectrometer time is spent J. Am. Chem. SOC. 1991, 113, 9354-9358 acquiring data rather than waiting for the spins to relax. Acknowledgment. It is a pleasure to thank Mr. David Koh for synthesizing DNA templates and Ms. Barbara Dengler for managing the laboratory.Abstract: To elucidate the three-dimensional solution structure of yeast copper-metallothionein (MT) from Saccharomyces cereuisiae, silver-substituted yeast metallothionein has been prepared as an isomorphic, NMR active, metal ion derivative. Using Ag-MT, iH-iwAg heteronuclear multiple quantum coherence transfer (HMQC) experiments have been performed to identify the individual ' "Ag resonances and their associated cysteine ligands, respectively. Specific factors associated with the optimized execution of the IH-lwAg HMQC experiments are identified and discussed. Analysis of the HMQC data has established the specific connectivities between I O of the 12 cysteine residues and seven bound IwAg(I) metal ions. The data confirm the exclusive involvement of cysteine thiolates in metal coordination and indicate that a minimum of eight cysteines are involved as bridging, shared ligands. Additionally, the present data suggest the existence of a mixed coordination number ( 2 and 3) for the seven bound Ag(1) ions.