Department of Biochemistry and Molecular Biophysics , Washington University in St. Louis , St. Louis , Missouri 63130 , United States.
Department of Medical Biotechnology and Translational Medicine , Università degli Studi di Milano , 20122 Milano , Italy.
J Phys Chem B. 2018 Dec 13;122(49):11468-11477. doi: 10.1021/acs.jpcb.8b07399. Epub 2018 Oct 1.
Quenching of the triplet state of tryptophan by close contact with cysteine provides a tool for measuring the rate of intramolecular contact formation, one of the most elementary events in the folding process, in peptides and proteins using only natural probes. Here we present a study performed on a stabilized mutant of the second β-hairpin of the GB1 domain, where we combine steady-state fluorescence, laser-induced temperature-jump, and contact formation measurements to unveil the role of elementary structural components on hairpin dynamics and overall stability. In particular, our methodology provides access to the conformational dynamics of both the folded and unfolded state of the hairpin under native conditions, revealing the presence of extremely slow dynamics on the microsecond time scale in the unfolded state and coexistence of structures with partial pairing of the tails in the folded state. Comparing model peptides that mimic the turn sequence, we found that both ion pairing and hydrogen bonding due to the threonine side chain contribute to the propensity of turn formation but not to the much slower dynamics of the hydrophobic core formation. Interestingly, the dynamics of the turn region in isolation are significantly faster than the dynamics measured for the unfolded state of the complete hairpin, suggesting that non-native hydrophobic contacts slow down the reconfiguration dynamics of the unfolded state. Overall, the information extracted from these experiments provides kinetic limits on interconversions among conformational populations, hence enabling a simplified multistate free-energy landscape for the GB1 hairpin to be drawn.
色氨酸与半胱氨酸的紧密接触猝灭三重态为测量肽和蛋白质中折叠过程最基本事件之一的分子内接触形成速率提供了一种工具，该过程仅使用天然探针。在这里，我们研究了 GB1 结构域第二个β发夹的稳定突变体，我们结合稳态荧光、激光诱导温度跃变和接触形成测量来揭示基本结构成分对发夹动力学和整体稳定性的作用。特别是，我们的方法可以在天然条件下访问发夹折叠和未折叠状态的构象动力学，揭示了未折叠状态下微秒时间尺度上极其缓慢的动力学以及折叠状态下尾部部分配对结构的共存。比较模拟转角序列的模型肽，我们发现由于苏氨酸侧链的离子对和氢键都有助于转角形成的倾向，但对疏水性核心形成的慢得多的动力学没有影响。有趣的是，孤立的转角区域的动力学明显快于完整发夹未折叠状态测量的动力学，这表明非天然疏水性接触会降低未折叠状态的重新配置动力学。总的来说，这些实验提取的信息为构象种群之间的转换提供了动力学限制，从而能够为 GB1 发夹绘制简化的多态自由能景观。
