Identification of the copper(II) coordinating residues in the prion protein by metal-catalyzed oxidation mass spectrometry: evidence for multiple isomers at low copper(II) loadings.
Identification of the copper(II) coordinating residues in the prion protein by metal-catalyzed oxidation mass spectrometry: evidence for multiple isomers at low copper(II) loadings.
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DOI：
10.1021/bi800970m
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发表时间：
2008-09-02
期刊：
影响因子：
2.9
通讯作者：
Vachet, Richard W.
中科院分区：
文献类型：
--
作者：
Srikanth, Rapole;Wilson, Jonathan;Burns, Colin S.;Vachet, Richard W.
虽然铜（II）的朊病毒蛋白的结合位点已被很好地研究在铜饱和条件下，身份的残基参与协调铜（II）在低化学计量和顺序，其中的结合位点负荷与铜（II），仍然没有得到解决。在这项研究中，我们已经使用了两种基于质谱的方法来收集不同化学计量负载的Cu（II）下的Cu（II）-朊病毒结合的洞察。第一种方法使用金属催化的氧化反应位点特异性修改的残基结合到Cu（II）在溶液中，和第二种方法确定Cu结合位点的基础上保护他的修改由焦碳酸二乙酯时，该残基结合Cu（II）在溶液中。对于这两种方法，通过这些反应标记的残基然后可以使用串联质谱法明确地鉴定。在将这两种互补方法应用于含有残基23-28和57-98的朊病毒蛋白的构建体时，得到了几个值得注意的观察结果。铜（II）的多个组氨酸咪唑的协调被发现在1：1和1：2的PrP：Cu（II）的比例。值得注意的是，在1：1复合物中似乎有四到七种这种多组氨酸配位模式的异构体。此外，我们的数据清楚地表明，His 96是主要的Cu（II）结合配体，因为在每个异构体中，His 96与Cu（II）结合。个别的octurepeat结合位点开始填充的比例为1：3的PrP：Cu（II），没有明确的偏好的顺序，他们加载Cu（II），虽然His 77 octurepeat似乎饱和最后。在低PrP：Cu（II）比率下存在几种“简并”Cu结合模式可允许其更容易地接受额外的Cu（II）离子，从而允许PrP作为细胞Cu（II）浓度的函数从单Cu（II）结合状态转变为多Cu（II）结合状态。
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.
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影响因子：
2.9
作者：
Burns, CS;Aronoff-Spencer, E;Millhauser, GL
通讯作者：
Millhauser, GL
影响因子：
4.1
作者：
Brown, DR;Wong, BS;Jones, IM
通讯作者：
Jones, IM
影响因子：
4.8
作者：
Morante, S;González-Iglesias, R;Gasset, M
通讯作者：
Gasset, M
影响因子：
4.8
作者：
Pauly, PC;Harris, DA
通讯作者：
Harris, DA
影响因子：
7.4
作者：
Bridgewater, JD;Vachet, RW
通讯作者：
Vachet, RW