Co-populated conformational ensembles of β2-microglobulin uncovered quantitatively by electrospray ionization mass spectrometry
Co-populated conformational ensembles of β2-microglobulin uncovered quantitatively by electrospray ionization mass spectrometry
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DOI：
10.1074/jbc.m401472200
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发表时间：
2004-06-25
影响因子：
4.8
通讯作者：
Ashcroft, AE
中科院分区：
文献类型：
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作者：
Borysik, AJH;Radford, SE;Ashcroft, AE
单个人β(2)-微球蛋白（β (2)m）有序组装成淀粉样蛋白原纤维与血液透析相关的淀粉样变性疾病有关。之前，我们已经证明，在酸性条件下（37℃下pH < 5.0），野生型β (2)m自发地组装成具有不同形态的原纤维。在这些条件下，β (2)m在体外具有许多不同的构象状态。然而，这种不同构象的平衡混合物很难用核磁共振或圆二色性等集合技术来解决。在这里，我们使用电喷雾电离质谱法来分析pH介于6.0和2.0之间的不同种类的β (2)m。我们表明，通过电荷态分布的线性反褶积，可以确定和量化每个构象系在整个pH范围内的分布程度。因此，在pH 3.6条件下，短原纤维的产生，构象系主要由以9+离子为中心的电荷态分布主导。相比之下，在酸性更强的条件下（pH 2.6），形成长而直的原纤维，电荷态分布以10+和11+离子为主。通过对β (2)m的两种变体（V9A和F30A）的研究，这些数据得到了加强，它们对pH变性的稳定性降低，并且显示出电荷态分布的pH依赖性的变化，这与色氨酸荧光测量的稳定性降低有关。这些数据强调了电喷雾电离质谱法在解决和量化不同构象物质的复杂混合物方面的潜力，其中一种或多种构象物质可能对淀粉样蛋白的形成很重要。
Ordered assembly of monomeric human beta(2)-microglobulin (beta(2)m) into amyloid fibrils is associated with the disorder hemodialysis-related amyloidosis. Previously, we have shown that under acidic conditions ( pH < 5.0 at 37 degrees C), wild-type beta(2)m assembles spontaneously into fibrils with different morphologies. Under these conditions, beta(2)m populates a number of different conformational states in vitro. However, this equilibrium mixture of conformationally different species is difficult to resolve using ensemble techniques such as nuclear magnetic resonance or circular dichroism. Here we use electrospray ionization mass spectrometry to resolve different species of beta(2)m populated between pH 6.0 and 2.0. We show that by linear deconvolution of the charge state distributions, the extent to which each conformational ensemble is populated throughout the pH range can be determined and quantified. Thus, at pH 3.6, conditions under which short fibrils are produced, the conformational ensemble is dominated by a charge state distribution centered on the 9+ ions. By contrast, under more acidic conditions ( pH 2.6), where long straight fibrils are formed, the charge state distribution is dominated by the 10+ and 11+ ions. The data are reinforced by investigations on two variants of beta(2)m (V9A and F30A) that have reduced stability to pH denaturation and show changes in the pH dependence of the charge state distribution that correlate with the decrease in stability measured by tryptophan fluorescence. The data highlight the potential of electrospray ionization mass spectrometry to resolve and quantify complex mixtures of different conformational species, one or more of which may be important in the formation of amyloid.