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

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Colin S. Burns (Creator)

Rapole Srikanth (Creator)

Richard W. Vachet (Creator)

Jonathan Wilson (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

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

Additional Information

Publication

Other

Biochemistry. 47:35(September 2008) p. 9258-9268.

Language: English

Date: 2011

Keywords

Cu(II) prion binding, different stoichiometric loadings, metal-catalyzed oxidation, diethyl pyrocarbonate

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