Consiglio Nazionale delle Ricerche, Istituto di Biofisica, 56124 Pisa, Italy.
J Phys Chem B. 2011 Nov 24;115(46):13755-64. doi: 10.1021/jp208397m. Epub 2011 Oct 28.
Using azurin as a model protein, this study enquires on the nature of small and large amplitude structural fluctuations permitting the penetration of different size solutes within protein folds, as inferred from quenching of the phosphorescence of buried Trp residues. The work examines the effect that guanidinium hydrochloride and urea have on the migration of a range of quencher molecules of increasing molecular size (M(w) range = 32-206 Da). Using the quenching rate constant of Trp phosphorescence as a monitor, the results demonstrate that structural fluctuations linked to O(2) migration are not affected by denaturants, whereas larger amplitude structural fluctuations necessary to facilitate penetration of bulkier quencher molecules [acrylonitrile, acrylamide, N-(hydroxymethyl) acrylamide, N-[tris(hydroxymethyl) methyl]acrylamide, and 2-acrylamido-2-methyl-1-propanesulfonic acid] are clearly enhanced by the presence of denaturant. These data when interpreted in the context of the amount of new protein surface uncovered to solvent (ΔSASA(o)) by the underlying structural fluctuations show a direct correlation between the amplitude of these motions and ΔSASA(o). Denaturants also promote slow frequency (20-80 s(-1)) conformational transitions, not manifested in normal aqueous solutions, which provide extra migration pathways for acrylamide and its larger derivatives. The quencher-size dependence of the quenching rate provides evidence of multiple, independent quencher migration pathways to the azurin core, which are characterized by motions on different time scales, microseconds and milliseconds, and by a 3- to 5-fold difference in ΔSASA(o), respectively.
本研究以天青蛋白为模型蛋白，通过猝灭埋藏色氨酸残基的磷光，探讨了允许不同大小溶质进入蛋白质折叠的小振幅和大振幅结构波动的性质。该工作考察了盐酸胍和尿素对一系列大小递增的猝灭剂分子（分子量范围=32-206 Da）迁移的影响。使用 Trp 磷光猝灭的速率常数作为监测指标，结果表明，与 O2 迁移相关的结构波动不受变性剂影响，而促进较大尺寸猝灭剂分子（丙烯腈、丙烯酰胺、N-羟甲基丙烯酰胺、N-[三（羟甲基）甲基]丙烯酰胺和 2-丙烯酰胺基-2-甲基-1-丙磺酸）进入的较大振幅结构波动则明显增强。这些数据在解释潜在结构波动所揭示的新蛋白质表面暴露于溶剂的程度（ΔSASA(o))时表明，这些运动的幅度与 ΔSASA(o)之间存在直接相关性。变性剂还促进了缓慢频率（20-80 s(-1))构象转变，在正常水溶液中未表现出这些转变，这为丙烯酰胺及其较大衍生物提供了额外的迁移途径。猝灭剂大小对猝灭速率的依赖性为天青蛋白核心提供了多个独立猝灭剂迁移途径的证据，这些途径的特征是不同时间尺度（微秒和毫秒）的运动以及 ΔSASA(o)分别相差 3-5 倍。
