Establishment of the metal-to-cysteine connectivities in silver-substituted yeast metallothionein

Abstract

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.

3D solution structure of copper and silver‐substituted yeast metallothioneins

“…One of the unresolved questions from earlier work concerned the anomalous chemical shift for metal ion VI which appeared experimentally to be only digonally coordinated but occurred in the chemical shift range of trigonally coordinated metal ions [6,7] suggesting the existence of a third ligand. Two candidates for this missing ligand to metal VI are sulphur ligands from Cys9 and/or Cys20, however, the final calculated structures did not reveal a preference for either of these Cys ligands in terms of an acceptable metal Vl-sulphur bond distance.…”

“…What tertiary structure exists can be attributed to the backbone twisting to accommodate the metal coordination with the deployment of the Cys in a fashion similar to the metal coordination in mammalian MT using Cys-XCys and Cys-XX-Cys structural motifs [24]. However, for yeast MT in contrast to the mammalian MTs, only half of the paired cysteine motifs (C-X-C) coordinate the same metal [6].…”

“…For the slowly exchanging amide protons whose acceptors could not be identified above, it is tempting to suggest that the acceptors might lie in the other half of a presumed dimer morphology as discussed below, There may be a fundamental difference in the coordination of metal II in CuMT and AgMT. Only two ligands have been identified for metal II for AgMT where Cys9 and Cysl 1 bind to different metals [6] and Cys9 is a terminal sulphur. It is conceivable that Cys9 may additionally be coordinated to metal II in CuMT, in which case successive coordination to metal II by Cys9 and Cysl 1 would necessitate the observed half turn in the backbone in the Q8-Cll segment, as in the mammalian Cys-X-Cys motif.…”

“…Distance and angle restraints were extracted from the reported experimental NMR data [6,7]. For AgMT, there are a total of 220 NOE constraints consisting of 60 intraresidue, 145 short range interresidue and 15 long range as summarized in Fig.…”

“…quantum coherence (HMQC) NMR experiments on the isomorphic, magnetically active (1 --1/2) Ag(I)-substituted yeast MT provided the requisite metal-to-Cys connectivities required for this protein's solution structure elucidation [5,6]. Additional structural constraints from NOE and coupling constant data for both the Ag(I) and the native Cu(I) protein were subsequently reported [7].…”