X-ray absorption spectroscopy is exquisitely sensitive to the coordination geometry of an absorbing atom and therefore allows bond distances and angles of the surrounding atomic cluster to be measured with atomic resolution. By contrast, the accuracy and resolution of metalloprotein active sites obtainable from x-ray crystallography are often insufficient to analyze the electronic properties of the metals that are essential for their biological functions. Here, we demonstrate that the combination of both methods on the same metalloprotein single crystal yields a structural model of the protein with exceptional active-site resolution. To this end, we have collected an x-ray diffraction data set to 1.4-angstrom resolution and Fe K-edge polarized x-ray absorption near edge structure (XANES) spectra on the same cyanomet sperm whale myoglobin crystal. The XANES spectra were quantitatively analyzed by using a method based on the multiple scattering approach, which yielded Fe-heme structural parameters with +/-(0.02-0.07)-angstrom accuracy on the atomic distances and +/- 7 degrees on the Fe-CN angle. These XANES-derived parameters were subsequently used as restraints in the crystal structure refinement. By combining XANES and x-ray diffraction, we have obtained an cyanomet sperm whale myoglobin structural model with a higher precision of the bond lengths and angles at the active site than would have been possible with crystallographic analysis alone.

X-ray structure analysis of a metalloprotein with enhanced active-site resolution using in situ x-ray absorption near edge structure spectroscopy / A., Arcovito; M., Benfatto; M., Cianci; S. S., Hasnain; K., Nienhaus; G. U., Nienhaus; C., Savino; R. W., Strange; Vallone, Beatrice; S., Della Longa. - In: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. - ISSN 0027-8424. - 104:15(2007), pp. 6211-6216. [10.1073/pnas.0608411104]

X-ray structure analysis of a metalloprotein with enhanced active-site resolution using in situ x-ray absorption near edge structure spectroscopy

VALLONE, Beatrice;
2007

Abstract

X-ray absorption spectroscopy is exquisitely sensitive to the coordination geometry of an absorbing atom and therefore allows bond distances and angles of the surrounding atomic cluster to be measured with atomic resolution. By contrast, the accuracy and resolution of metalloprotein active sites obtainable from x-ray crystallography are often insufficient to analyze the electronic properties of the metals that are essential for their biological functions. Here, we demonstrate that the combination of both methods on the same metalloprotein single crystal yields a structural model of the protein with exceptional active-site resolution. To this end, we have collected an x-ray diffraction data set to 1.4-angstrom resolution and Fe K-edge polarized x-ray absorption near edge structure (XANES) spectra on the same cyanomet sperm whale myoglobin crystal. The XANES spectra were quantitatively analyzed by using a method based on the multiple scattering approach, which yielded Fe-heme structural parameters with +/-(0.02-0.07)-angstrom accuracy on the atomic distances and +/- 7 degrees on the Fe-CN angle. These XANES-derived parameters were subsequently used as restraints in the crystal structure refinement. By combining XANES and x-ray diffraction, we have obtained an cyanomet sperm whale myoglobin structural model with a higher precision of the bond lengths and angles at the active site than would have been possible with crystallographic analysis alone.
2007
crystallographic restraints; exafs; myoglobin; protein crystallography; synchroton radiation; xanes
01 Pubblicazione su rivista::01a Articolo in rivista
X-ray structure analysis of a metalloprotein with enhanced active-site resolution using in situ x-ray absorption near edge structure spectroscopy / A., Arcovito; M., Benfatto; M., Cianci; S. S., Hasnain; K., Nienhaus; G. U., Nienhaus; C., Savino; R. W., Strange; Vallone, Beatrice; S., Della Longa. - In: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. - ISSN 0027-8424. - 104:15(2007), pp. 6211-6216. [10.1073/pnas.0608411104]
File allegati a questo prodotto
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/68791
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? 12
  • Scopus 64
  • ???jsp.display-item.citation.isi??? 63
social impact