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

Abstract

The 600-MHz 'H-NMR NOESY spectra on Co(II),-reconstituted metallothionein (Co,MT), 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 'H-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 C04S1 cluster of Co,MT. Together with results from one-dimensional NOE experiments, the two-dimensional experiments allowed us to proceed with the painvise assignment of the isotropically shifted signals of the CBH2 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 propose sequence-specific assignments of a few hyperfine-shifted 'H-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 C O~S~ cluster are drawn.Metallothioneins (MT) are storage proteins, rich in cysteine which can bind up to seven zinc or cadmium atoms in two clusters [l]. One cluster is formed by four metal ions which are bound to eleven cysteines (M4S11) and the other by three metal ions and nine cysteines (M3S9). We have recently reported on the 'H-NMR properties of Co(II), rabbit liver metallothionein (Co7MT) [2,3]. Co7MT gives rise to a surprisingly well-resolved 'H-NMR spectrum characterized by more than 20 isotropically shifted signals spread between 350 ppm and -50 ppm, with very short longitudinal (T,) and transverse ( T z ) nuclear-relaxation times. The signals have been assigned to the C04Sl1 cluster domain, by comparing the spectra of the Co,MT and the Co3Cd4MT derivatives [2]. Most of the far isotropically shifted 'H-NMR signals are believed to arise from the C j H z protons of the eleven cysteines involved in metal coordination, experiencing large contact contributions; some of the less-shifted signals, both up and downfield, could belong to Cys CcrH protons.To proceed with the assignment of the more than 20 far isotropically shifted 'H-NMR signals, additional information on the system is needed. In the last decade, in spite of technical difficulties, a number of one-dimensional (1 D) [4 - Thanks to the activity of a few research groups, 2D NOE spectroscopy (NOESY) techniques have been progressively applied to paramagnetic metalloproteins of increasing difficulty, from low-spin Fe(II1) heme proteins featuring chemicalshift ranges of about 30ppm and short Tl of about 10ms 161, heme proteins [17], Co/Cu superoxide dismutase [18], Co(I1)-substituted metalloenzymes [19], and so on. We report ...

Coordination of three and four Cu(I) to the α− and β‐domain of vertebrate Zn‐metallothionein‐1, respectively, induces significant structural changes

“…The spectra of both the only Zn(II)-containing b-domain and its Cu(I) 4 derivative seem to be of lower quality with large areas of broad overlapping peaks. This behaviour might be due to higher flexibility within the b-domain which has been reported already before [17][18][19][20]. TOCSY spectra (not Table 1.…”

“…The spectra of both the only Zn(II)‐containing β‐domain and its Cu(I) 4 derivative seem to be of lower quality with large areas of broad overlapping peaks. This behaviour might be due to higher flexibility within the β‐domain which has been reported already before [17–20]. TOCSY spectra (not shown) were recorded for the Zn x Cu 3 αMT‐1 and Zn y Cu 4 βMT‐1 derivatives and used, together with the NOESY spectra, to obtain their sequence specific assignments (supplementary Tables S1 and S2).…”

“…Comparative NMR studies provided evidence that Zn(II) can isomorphically replace Cd(II) in MT‐2 [16]. This result was corroborated by NMR studies on cobalt substituted MTs, as cobalt was often used as a zinc analogue in structural investigations [17–19]. The solution structure of murine 113 Cd 7 MT‐1 showed high similarity with rat liver MT‐2.…”

Metal coordination of azurin in the unfolded state

“…Signal A integrates as two protons and shows a chemical shift of 174 ppm. This value is typical for CHβ protons belonging to cysteine ligands not only in cobalt(II) [25, 32–34]but also in nickel(II)‐substituted proteins [29, 35, 36], as well as in iron‐sulphur proteins [37–39]. As in the native protein (Fig.…”

The Bioinorganic Chemistry of Mammalian Metallothioneins

“…184−186 Spectra of Co(II) complexes allow a direct observation of d-d bands and conclusions about coordination environment and geometries of the metal ions in partially and fully saturated MT species. 133,187 Fluorimetric methods were applied for structural studies of MT with attached FRET probes and to examine the metal association and dissociation and the determination of metal affinities. 73,151,154,188,189 113 Cd and 111 Cd-NMR spectroscopy was critical for the elucidation of the cluster formation in the αand β-domains, cluster dynamics, and the kinetics of Cd(II) transfer.…”