Structure Of Metal-binding Sites Research Articles

Interaction of Cd and Zn with biologically important ligands characterized using solid-state NMR and ab initio calculations.

For the first time, coordination geometry and structure of metal binding sites in biologically relevant systems are studied using chemical shift parameters obtained from solid-state NMR experiments and quantum chemical calculations. It is also the first extensive report looking at metal-imidazole interaction in the solid state. The principal values of the (113)Cd chemical shift anisotropy (CSA) tensor in crystalline cadmium histidinate and two different cadmium formates (hydrate and anhydrate) were experimentally measured to understand the effect of coordination number and geometry on (113)Cd CSA. Further, (13)C and (15)N chemical shifts have also been experimentally determined to examine the influence of cadmium on the chemical shifts of (15)N and (13)C nuclei present near the metal site in the cadmium-histidine complex. These values were then compared with the chemical shift values obtained from the isostructural bis(histidinato)zinc(II) complex as well as from the unbound histidine. The results show that the isotropic chemical shift values of the carboxyl carbons shift downfield and those of amino and imidazolic nitrogens shift upfield in the metal (Zn,Cd)-histidine complexes relative to the values of the unbound histidine sample. These shifts are in correspondence with the anticipated values based on the crystal structure. Ab initio calculations on the cadmium histidinate molecule show good agreement with the (113)Cd CSA tensors determined from solid-state NMR experiments on powder samples. (15)N chemical shifts for other model complexes, namely, zinc glycinate and zinc hexaimidazole chloride, are also considered to comprehend the effect of zinc binding on (15)N chemical shifts.

Use of near-infrared Yb 3+ vibronic side band spectroscopy to probe protein metal-binding sites

Near infrared Yb 3+ vibronic sideband spectroscopy is used to obtain structural information of metal binding sites in metalloproteins. This novel technique, using near-infrared excitation and detection (970–1200 nm) provides a selective well-resolved “infrared-like” vibrational spectrum of those ligands which are chelated to the Yb 3+ ion. Weak vibronic sidebands (VSB) whose energy shifts, with respect to the main 2 F 5 2 → 2 F 7 2 Yb 3+ zero-phonon electronic transition, represent the vibrational frequencies of the Yb 3+ ligands. As a model complex, we have studied the VSB spectrum of Yb 3+ complexed with Eriochrome Cyanine R (Yb:ECR), a ligand which possesses COO −, CO, and CO functional groups. VSB arising from the vibrational modes of these chemical groups are readily observed and well resolved. VSB fluorescence spectra of Yb 3+ reconstituted into the Fe 3+ binding sites of the iron-transporting protein human serum transferrin indicates that these two sites are not spectroscopically identical. The chemical nature of the ligands of the Yb 3+ in this protein, as deduced by the observed VSB frequencies, are entirely in agreement with the known crystal structure. This technique demonstrates enormous potential in elucidating the localized structure of metal binding sites in proteins.

Direct vibrational structure of protein metal-binding sites from near-infrared Yb3+ vibronic side band spectroscopy.

Near-infrared Yb3+ vibronic side band (VSB) spectroscopy is used to obtain structural information of metal binding sites in metalloproteins. This technique provides a selective "IR-like" vibrational spectrum of those ligands chelated to the Yb3+ ion. VSB spectra of various model complexes of Yb3+ representing different ligand types were studied to provide references for the VSB spectra of Yb(3+)-reconstituted metalloproteins. Ca2+ in the calcium-binding protein parvalbumin and Fe3+ in the iron-transporting protein transferrin were replaced with Yb3+. The fluorescence of Yb3+ reconstituted into these two proteins exhibits weak VSBs whose energy shifts, with respect to the main 2F5/2-->2F7/2 Yb3+ electronic transition, represent the vibrational frequencies of the Yb3+ ligands. The chemical nature of the ligands of the Yb3+ in these proteins, as deduced by the observed VSB frequencies, is entirely in agreement with their known crystal structures. For transferrin, replacement of the 12CO3(2-) metal counterion with 13CO3(2-) yielded the expected isotopic shift for the VSBs corresponding to the carbonate vibrational modes. This technique demonstrates enormous potential in elucidating the localized structure of metal binding sites in proteins.