Department of Chemistry, University of Massachusetts Amherst, Amherst, Massachusetts 01003, United States.
Anal Chem. 2020 Apr 7;92(7):4731-4735. doi: 10.1021/acs.analchem.0c00368. Epub 2020 Mar 17.
Obtaining kinetic and thermodynamic information for protein amyloid formation can yield new insight into the mechanistic details of this biomedically important process. The kinetics of the structural change that initiates the amyloid pathway, however, has been challenging to access for any amyloid protein system. Here, using the protein β-2-microglobulin (β2m) as a model, we measure the kinetics and energy barrier associated with an initial amyloidogenic structural change. Using covalent labeling and mass spectrometry, we measure the decrease in solvent accessibility of one of β2m's Trp residues, which is buried during the initial structural change, as a way to probe the kinetics of this structural change at different temperatures and under different amyloid forming conditions. Our results provide the first-ever measure of the activation barrier for a structural change that initiates the amyloid formation pathway. The results also yield new mechanistic insight into β2m's amyloidogenic structural change, especially the role of Pro32 isomerization in this reaction.
获得蛋白质淀粉样形成的动力学和热力学信息可以深入了解这一具有重要医学意义的过程的机制细节。然而，对于任何淀粉样蛋白体系，起始淀粉样途径的结构变化的动力学一直难以获得。在这里，我们使用蛋白 β-2-微球蛋白 (β2m) 作为模型，测量与初始淀粉样形成结构变化相关的动力学和能量障碍。使用共价标记和质谱法，我们测量β2m 的一个色氨酸残基的溶剂可及性降低，该残基在初始结构变化过程中被埋藏，以此在不同温度和不同淀粉样形成条件下探测该结构变化的动力学。我们的结果首次提供了起始淀粉样形成途径的结构变化的活化能垒的测量值。结果还为β2m 的淀粉样形成结构变化提供了新的机制见解，特别是脯氨酸 32 异构化在该反应中的作用。
