Department of Biological Sciences, Indian Institute of Science Education and Research (IISER), Mohali, Knowledge City, Sector 81, S.A.S. Nagar, Mohali, 140306, India.
Eur Biophys J. 2013 Oct;42(10):767-76. doi: 10.1007/s00249-013-0923-z. Epub 2013 Aug 24.
Human β2-microglobulin (β2m) aggregation is implicated in dialysis-related amyloidosis. Previously, it has been shown that β2m adopts an ensemble of partially unfolded states at low pH. Here we provide detailed structural and dynamical insights into the acid unfolded and yet compact state of β2m at pH 2.5 using a host of fluorescence spectroscopic tools. These tools allowed us to investigate protein conformational dynamics at low micromolar protein concentrations in an amyloid-forming condition. Our equilibrium fluorescence data in combination with circular dichroism data provide support in favor of progressive structural dissolution of β2m with lowering pH. The acid unfolded intermediate at pH 2.5 has high 8-anilinonaphthalene, 1-sulfonic acid (ANS)-binding affinity and is devoid of significant secondary structural elements. Using fluorescence lifetime measurements, we have been able to monitor the conformational transition during the pH transition from the native to the compact disordered state. Additionally, using time-resolved fluorescence anisotropy measurements, we have been able to distinguish this compact disordered state from the canonical denatured state of the protein by identifying unique dynamic signatures pertaining to the segmental chain mobility. Taken together, our results demonstrate that β2m at pH 2.5 adopts a compact noncanonical unfolded state resembling a collapsed premolten globule state. Additionally, our stopped-flow fluorescence kinetics results provide mechanistic insights into the formation of a compact disordered state from the native form.
人β2-微球蛋白（β2m）聚集与透析相关淀粉样变性有关。先前已经表明，β2m 在低 pH 值下采用部分展开的状态集合。在这里，我们使用一系列荧光光谱工具详细研究了β2m 在 pH 2.5 时的酸展开但仍紧凑的状态的结构和动力学。这些工具使我们能够在形成淀粉样蛋白的条件下，在低微摩尔蛋白浓度下研究蛋白质构象动力学。我们的平衡荧光数据与圆二色性数据相结合，为β2m 随 pH 值降低而逐渐结构溶解提供了支持。在 pH 2.5 时的酸展开中间物具有高 8-苯胺萘-1-磺酸（ANS）结合亲和力，并且没有明显的二级结构元件。使用荧光寿命测量，我们能够监测从天然到紧凑无序状态的 pH 转换过程中的构象转变。此外，通过使用时间分辨荧光各向异性测量，我们能够通过识别与分段链流动性有关的独特动态特征，将这种紧凑的无序状态与蛋白质的典型变性状态区分开来。总之，我们的结果表明，β2m 在 pH 2.5 时采用类似于塌陷预熔球状态的紧凑非典型展开状态。此外，我们的停流荧光动力学结果为从天然形式形成紧凑无序状态提供了机制见解。
