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.
Roselli
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C
Section de Bioénergétique, CEA, Gif-sur-Yvette, France.
Boussac
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A
Mattioli
T A
TA
D001059:Apoproteins / Q000737:chemistry; D001665:Binding Sites; D008024:Ligands; D008667:Metalloproteins / Q000737:chemistry*; D008674:Metals, Rare Earth; D010320:Parvalbumins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013055:Spectrophotometry, Infrared; D014168:Transferrin / Q000737:chemistry*; D015018:Ytterbium / Q000737:chemistry*
D001059:Apoproteins; D008024:Ligands; D008667:Metalloproteins; D008674:Metals, Rare Earth; D010320:Parvalbumins; D014168:Transferrin; D015018:Ytterbium