Astbury Centre for Structural Molecular Biology, Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK.
Rapid Commun Mass Spectrom. 2012 Aug 30;26(16):1783-92. doi: 10.1002/rcm.6282.
Amyloid formation is implicated in a number of human diseases. β(2)-Microglobulin (β(2)m) is the precursor protein in dialysis-related amyloidosis and it has been shown that partial, or more complete, unfolding is key to amyloid fibril formation in this pathology. Here the relationship between conformational flexibility and β(2)m amyloid formation at physiological pH has been investigated.
HDX-ESI-MS was used to study the conformational dynamics of β(2)m. Protein engineering, or the addition of Cu(2+) ions, sodium dodecyl sulphate, trifluoroethanol, heparin, or protein stabilisers, was employed to perturb the conformational dynamics of β(2)m. The fibril-forming propensities of the protein variants and the wild-type protein in the presence of additives, which resulted in >5-fold increase in the EX1 rate of HDX, were investigated further.
ESI-MS revealed that HDX occurs via a mixed EX1/EX2 mechanism under all conditions. Urea denaturation and tryptophan fluorescence indicated that EX1 exchange occurred from a globally unfolded state in wild-type β(2)m. Although >30-fold increase in the HDX exchange rate was observed both for the protein variants and for the wild-type protein in the presence of specific additives, large increases in exchange rate did not necessarily result in extensive de novo fibril formation.
The conformational dynamics measured by the EX1 rate of HDX do not predict the ability of β(2)m to form amyloid fibrils de novo at neutral pH. This suggests that the formation of amyloid fibrils from β(2)m at neutral pH is dependent on the generation of one or more specific aggregation-competent species which facilitate self-assembly.
淀粉样变的形成与许多人类疾病有关。β(2)-微球蛋白（β(2)m）是透析相关性淀粉样变的前体蛋白，已有研究表明，部分或更完全的展开是该病理中淀粉样纤维形成的关键。本研究旨在探讨生理 pH 下构象灵活性与β(2)m 淀粉样形成之间的关系。
使用 HDX-ESI-MS 研究β(2)m 的构象动力学。采用蛋白质工程或添加 Cu(2+)离子、十二烷基硫酸钠、三氟乙醇、肝素或蛋白质稳定剂等方法，干扰β(2)m 的构象动力学。进一步研究了在添加剂存在下，蛋白变体和野生型蛋白的纤维形成倾向，这些添加剂导致 HDX 的 EX1 速率增加了 5 倍以上。
ESI-MS 表明，在所有条件下，HDX 均通过混合 EX1/EX2 机制发生。脲变性和色氨酸荧光表明，野生型β(2)m 中 EX1 交换发生在整体展开状态。尽管在特定添加剂存在下，蛋白变体和野生型蛋白的 HDX 交换速率均增加了 30 倍以上，但交换速率的大幅增加并不一定导致广泛的从头纤维形成。
通过 HDX 的 EX1 速率测量的构象动力学并不能预测β(2)m 在中性 pH 下从头形成淀粉样纤维的能力。这表明，β(2)m 在中性 pH 下形成淀粉样纤维依赖于一种或多种特定的聚集能力物种的产生，这些物种促进了自组装。
