Abstract
In this work we present and analyse XAS measurements carried out on various portions of Prion-protein tetra-octa-repeat peptides in complexes with Cu(II) ions, both in the presence and in the absence of Zn(II). Because of the ability of the XAS technique to provide detailed local structural information, we are able to demonstrate that Zn acts by directly interacting with the peptide, in this way competing with Cu for binding with histidine. This finding suggests that metal binding competition can be important in the more general context of metal homeostasis.
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Notes
For a more detailed description of how the χ(k) EXAFS signal is extracted from XAS data see the Appendix in Minicozzi et al. (2008).
The normalization is performed in a standard way using the ATHENA software (Ravel 2008), i.e. fitting the pre-edge region with a straight line and subtracting it from the whole spectrum (Ravel and Newville 2005). The post-edge region data are then fitted with a polynomial. Finally, the jump between the pre-edge and the post-edge fits at the edge energy is calculated and the spectrum is divided by the height of the jump. We recall the standard formulae \( {\chi (k) = \frac{{\mu (E) - \mu_{0} (E)}}{{\mu_{0} (E)}}} \) and \( {\hbar k = \sqrt {2m(E - E_{0} )} } \) where μ 0(E) is the single atom absorption coefficient and E 0 is the edge energy.
For completeness we provide in the supplementary material a quantitative analysis of the EXAFS spectra of samples S3 and S3_Zn data at the Cu K-edge. The analysis confirms that the structure of the Cu2+ binding site is not affected by the presence of Zn2+ and the fitted site geometry is highly consistent with the available crystallographic information (Chattopadhyay et al. 2005).
It should be said that here we have simplified the analysis by assuming that component β corresponds to a situation in which all four His units of each tetra-octa-repeat are simultaneously involved in binding the metal. For the sake of completeness, the same analysis has also been performed assuming that Cu2+ in component β is bound to three His units. The results obtained under this assumption do not differ from those presented here in the text and can be found in the supplementary material.
Here we make the further hypothesis that the reason why Zn2+ can be found free in solution it is that there are no more available His units.
The quality factor R is computed by use of the formula: \( {\text{R}} = \sum\nolimits_{{{\text{i}} = 1}}^{\text{P}} {\frac{ 1}{{{\text{w}}_{\text{i}} }}} \left| {{{\upchi}}^{ \exp } (k_{\text{i}} )- {{\upchi}}^{\text{fit}} (k_{\text{i}} )} \right| \) where P is the number of experimental points and \( {\text{w}}_{\text{i}} = \frac{1}{{k_{i}^{n} }}\sum\nolimits_{j = 1}^{P} {k_{j}^{n} |{{\upchi}}^{\exp } (k_{j} )| \, } \) in which n is an integer which is normally taken to lie between 0 and 3 (here we took n = 3).
In all the fits only the distance and relative position of the nearest neighbouring atoms in the His-bound Zn2+ environment are taken as free variables.
Incidentally, we have also tried to leave the Zn2+ fraction in buffer as a free variable in the fit to the S1_Zn XAS spectra (data not shown). Values in good agreement with the numbers in the second column of Table 2 are obtained when the structural models M1 and/or M2 are assumed. Completely unrealistic values are instead selected by the fit with models M3 and M4.
In the supplementary material the same plot as in Fig. 9 is drawn by also taking into account the errors in the 〈r in〉 determination. In this way both M1 and M2 models yield numbers for 〈r in〉 that fall within the range of the acceptable BVS values as defined in the text.
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Acknowledgments
We are very grateful to G.C. Rossi for useful discussions and for reading the manuscript. Partial financial support from PRIN08 is acknowledged. We thank the anonymous referees for their useful suggestions.
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F. Stellato, A. Spevacek contributed equally to the work.
Special Issue: SIBPA Meeting 2011.
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Stellato, F., Spevacek, A., Proux, O. et al. Zinc modulates copper coordination mode in prion protein octa-repeat subdomains. Eur Biophys J 40, 1259–1270 (2011). https://doi.org/10.1007/s00249-011-0713-4
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DOI: https://doi.org/10.1007/s00249-011-0713-4