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The correlation of 113Cd NMR and 111mCd PAC spectroscopies provides a powerful approach for the characterization of the structure of Cd(II)-substituted Zn(II) proteins.

ABSTRACT: Cd(II) has been used as a probe of zinc metalloenzymes and proteins because of the spectroscopic silence of Zn(II). One of the most commonly used spectroscopic techniques is (113)Cd NMR; however, in recent years (111m)Cd Perturbed Angular Correlation spectroscopy ((111m)Cd PAC) has also been shown to provide useful structural, speciation and dynamics information on Cd(II) complexes and biomolecules. In this article, we show how the joint use of (113)Cd NMR and (111m)Cd PAC spectroscopies can provide detailed information about the Cd(II) environment in thiolate-rich proteins. Specifically we show that the (113)Cd NMR chemical shifts observed for Cd(II) in the designed TRI series (TRI = Ac-G(LKALEEK)(4)G-NH(2)) of peptides vary depending on the proportion of trigonal planar CdS(3) and pseudotetrahedral CdS(3)O species present in the equilibrium mixture. PAC spectra are able to quantify these mixtures. When one compares the chemical shift range for these peptides (from delta = 570 to 700 ppm), it is observed that CdS(3) species have delta 675-700 ppm, CdS(3)O complexes fall in the range delta 570-600 ppm and mixtures of these forms fall linearly between these extremes. If one then determines the pK(a2) values for Cd(II) complexation [pK(a2) is for the reaction Cd[(peptide-H)(2)(peptide)](+)-->Cd(peptide)(3)(-) + 2H(+)] and compares these to the observed chemical shift for the Cd(peptide)(3)(-) complexes, one finds that there is also a direct linear correlation. Thus, by determining the chemical shift value of these species, one can directly assess the metal-binding affinity of the construct. This illustrates how proteins may be able to fine tune metal-binding affinity by destabilizing one metallospecies with respect to another. More important, these studies demonstrate that one may have a broad (113)Cd NMR chemical shift range for a chemical species (e.g., CdS(3)O) which is not necessarily a reflection of the structural diversity within such a four-coordinate species, but rather a consequence of a fast exchange equilibrium between two related species (e.g., CdS(3)O and CdS(3)). This could lead to reinterpretation of the assignments of cadmium-protein complexes and may impact the application of Cd(II) as a probe of Zn(II) sites in biology.

SUBMITTER: Iranzo O

PROVIDER: S-EPMC3598615 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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The correlation of 113Cd NMR and 111mCd PAC spectroscopies provides a powerful approach for the characterization of the structure of Cd(II)-substituted Zn(II) proteins.

Iranzo Olga O Jakusch Tamas T Lee Kyung-Hoon KH Hemmingsen Lars L Pecoraro Vincent L VL

Chemistry (Weinheim an der Bergstrasse, Germany) 20090101 15

Cd(II) has been used as a probe of zinc metalloenzymes and proteins because of the spectroscopic silence of Zn(II). One of the most commonly used spectroscopic techniques is (113)Cd NMR; however, in recent years (111m)Cd Perturbed Angular Correlation spectroscopy ((111m)Cd PAC) has also been shown to provide useful structural, speciation and dynamics information on Cd(II) complexes and biomolecules. In this article, we show how the joint use of (113)Cd NMR and (111m)Cd PAC spectroscopies can pro ...[more]

PMID: 19229934

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Project description:A minigene encoding the C-terminal domain of the 2Fe rubredoxin of Pseudomonas oleovorans was created from the parental alk G gene contained in the expression plasmid pKK223-3. The vector directed the high-level production of the C-terminal domain of this rubredoxin; a simple procedure was used to purify the recombinant domain in the 1Fe form. The 1Fe form of the C-terminal domain was readily converted into the apoprotein and cadmium forms after precipitation with trichloroacetic acid and resolubilization in the presence or absence of cadmium chloride respectively. In steady-state assays, the recombinant 1Fe C-terminal domain is redox-active and able to transfer electrons from reduced rubredoxin reductase to cytochrome c. The absorption spectrum and dichroic features of the CD spectrum for the iron- and cadmium-substituted C-terminal domain are similar to those reported for the iron- and cadmium-substituted Desulfovibrio gigas rubredoxin [Henehen, Pountney, Zerbe and Vasak (1993) Protein Sci. 2, 1756-1764]. Difference absorption spectroscopy of the cadmium-substituted C-terminal domain revealed the presence of four Gaussian-resolved maxima at 202, 225, 240 and 276 nm; from Jørgensen's electronegativity theory, the 240 nm band is attributable to a CysS-Cd(II) charge-transfer excitation. Attempts to express the N-terminal domain of the 2Fe rubredoxin directly from a minigene were unsuccessful. However, the N-terminal domain was isolated through cleavage of an engineered 2Fe rubredoxin in which a factor Xa proteolysis site had been introduced into the putative interdomain linker. The N-terminal domain is characterized by absorption spectra typical of the 1Fe rubredoxins. The domain is folded as determined by CD and NMR spectroscopies and is redox-active. However, the N-terminal domain is less stable than the isolated C-terminal domain, a finding consistent with the known properties of the full-length 2Fe and cadmium-substituted Ps. oleovorans rubredoxin.

Dataset Information

The correlation of 113Cd NMR and 111mCd PAC spectroscopies provides a powerful approach for the characterization of the structure of Cd(II)-substituted Zn(II) proteins.

Publications

The correlation of 113Cd NMR and 111mCd PAC spectroscopies provides a powerful approach for the characterization of the structure of Cd(II)-substituted Zn(II) proteins.

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