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    Identification of the Cu(II) Coordinating Residues in the Prion Protein by Metal-Catalyzed Oxidation Mass Spectrometry: Evidence for Multiple Isomers at Low Cu(II) Loadings

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    Author
    Srikanth, Rapole; Wilson, Jonathan; Burns, Colin Sanderson; Vachet, Richard W.
    Abstract
    While the Cu(II) binding sites of the prion protein have been well studied under Cu-saturation conditions, the identity of the residues involved in coordinating Cu(II) at low stoichiometries and the order in which the binding sites load with Cu(II), remain unresolved. In this study, we have used two mass spectrometry based methods to gather insight into Cu(II)-prion binding under different stoichiometric loadings of Cu(II). The first method uses metal-catalyzed oxidation reactions to site specifically modify the residues bound to Cu(II) in solution, and the second method determines Cu binding sites based on the protection of His from modification by diethyl pyrocarbonate when this residue binds Cu(II) in solution. For both methods, the residues that are labeled by these reactions can then be unambiguously identified using tandem mass spectrometry. Upon applying these two complementary methods to a construct of the prion protein that contains residues 23-28 and 57-98, several noteworthy observations are made. Coordination of Cu(II) by multiple His imidazoles is found at 1:1 and 1:2 PrP:Cu(II) ratios. Notably, there appear to be four to seven isomers of this multiple histidine coordination mode in the 1:1 complex. Furthermore, our data clearly show that His96 is the dominant Cu(II) binding ligand, as in every isomer His96 is bound to Cu(II). The individual octarepeat binding sites begin to fill at ratios of 1:3 PrP:Cu(II) with no clear preference for the order in which they load with Cu(II), although the His77 octarepeat appears to saturate last. The existence of several ‘degenerate’ Cu binding modes at low PrP:Cu(II) ratios may allow it to more readily accept additional Cu(II) ions, thus allowing PrP to transition from a singly Cu(II) bound state to a multiply Cu(II) bound state as a function of cellular Cu(II) concentration. Originally published Biochemistry, Vol. 47, No. 35, Sep 2008
    URI
    http://hdl.handle.net/10342/3419
    Subject
     Cu(II) prion binding; Different stoichiometric loadings; Metal-catalyzed oxidation; Diethyl pyrocarbonate 
    Date
    2008-09-02
    Citation:
    APA:
    Srikanth, Rapole, & Wilson, Jonathan, & Burns, Colin Sanderson, & Vachet, Richard W.. (September 2008). Identification of the Cu(II) Coordinating Residues in the Prion Protein by Metal-Catalyzed Oxidation Mass Spectrometry: Evidence for Multiple Isomers at Low Cu(II) Loadings. Biochemistry, (47:35), p.9258-9268. Retrieved from http://hdl.handle.net/10342/3419

    Display/Hide MLA, Chicago and APA citation formats.

    MLA:
    Srikanth, Rapole, and Wilson, Jonathan, and Burns, Colin Sanderson, and Vachet, Richard W.. "Identification of the Cu(II) Coordinating Residues in the Prion Protein by Metal-Catalyzed Oxidation Mass Spectrometry: Evidence for Multiple Isomers at Low Cu(II) Loadings". Biochemistry. 47:35. (9258-9268.), September 2008. March 07, 2021. http://hdl.handle.net/10342/3419.
    Chicago:
    Srikanth, Rapole and Wilson, Jonathan and Burns, Colin Sanderson and Vachet, Richard W., "Identification of the Cu(II) Coordinating Residues in the Prion Protein by Metal-Catalyzed Oxidation Mass Spectrometry: Evidence for Multiple Isomers at Low Cu(II) Loadings," Biochemistry 47, no. 35 (September 2008), http://hdl.handle.net/10342/3419 (accessed March 07, 2021).
    AMA:
    Srikanth, Rapole, Wilson, Jonathan, Burns, Colin Sanderson, Vachet, Richard W.. Identification of the Cu(II) Coordinating Residues in the Prion Protein by Metal-Catalyzed Oxidation Mass Spectrometry: Evidence for Multiple Isomers at Low Cu(II) Loadings. Biochemistry. September 2008; 47(35) 9258-9268. http://hdl.handle.net/10342/3419. Accessed March 07, 2021.
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    • Chemistry
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    East Carolina University

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