Journal
CHEMISTRY-A EUROPEAN JOURNAL
Volume 15, Issue 15, Pages 3761-3772Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/chem.200802105
Keywords
cadmium; metallopeptides; NMR spectroscopy; zinc proteins
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Funding
- National Institute of Health [R01 ESO 12236]
- Margaret and Herman Sokol Foundation
- The Danish Natural Science Research Council
- NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES [R01ES012236] Funding Source: NIH RePORTER
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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 Cd-113 NMR; however, in recent years Cd-111m Perturbed Angular Correlation spectroscopy (Cd-111m PAC) has also been shown to provide useful structural, speciation and dynamics information oil Cd-II complexes and biomolecules. In this article, we show how the joint use of Cd-113 NMR and Cd-111m PAC spectroscopies can provide detailed information about the Cd-II environment in thiolate-rich proteins. Specifically we show that the Cd-113 NMR chemical shifts observed for Cd-II in the designed TRI series (TRI = Ac-G-(LKALEEK)(4)G-NH2) of peptides vary depending on the proportion of trigonal planar CdS3 and pseudotetrahedral CdS3O 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 CdS3 species have delta 675-700 ppm, CdS3O 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 Cd-113 NMR chemical shift range for a chemical species (e.g., CdS3O) 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., CdS3O and CdS3). 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.
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