CQC, Department of Chemistry , University of Coimbra , 3004-535 Coimbra , Portugal.
J Phys Chem B. 2018 Apr 12;122(14):3790-3800. doi: 10.1021/acs.jpcb.8b00229. Epub 2018 Mar 30.
The understanding of fast folding dynamics of single α-helices comes mostly from studies on rationally designed peptides displaying sequences with high helical propensity. The folding/unfolding dynamics and energetics of α-helix conformations in naturally occurring peptides remains largely unexplored. Here we report the study of a protein fragment analogue of the C-peptide from bovine pancreatic ribonuclease-A, RN80, a 13-amino acid residue peptide that adopts a highly populated helical conformation in aqueous solution. H NMR and CD structural studies of RN80 showed that α-helix formation displays a pH-dependent bell-shaped curve, with a maximum near pH 5, and a large decrease in helical content in alkaline pH. The main forces stabilizing this short α-helix were identified as a salt bridge formed between Glu-2 and Arg-10 and the cation-π interaction involving Tyr-8 and His-12. Thus, deprotonation of Glu-2 or protonation of His-12 are essential for the RN80 α-helix stability. In the present study, RN80 folding and unfolding were triggered by laser-induced pH jumps and detected by time-resolved photoacoustic calorimetry (PAC). The photoacid proton release, amino acid residue protonation, and unfolding/folding events occur at different time scales and were clearly distinguished using time-resolved PAC. The partial unfolding of the RN80 α-helix, due to protonation of Glu-2 and consequent breaking of the stabilizing salt bridge between Glu-2 and Arg-10, is characterized by a concentration-independent volume expansion in the sub-microsecond time range (0.8 mL mol, 369 ns). This small volume expansion reports the cost of peptide backbone rehydration upon disruption of a solvent-exposed salt bridge, as well as backbone intrinsic expansion. On the other hand, RN80 α-helix folding triggered by His-12 protonation and subsequent formation of a cation-π interaction leads to a microsecond volume contraction (-6.0 mL mol, ∼1.7 μs). The essential role of two discrete side chain interactions, a salt bridge, and in particular a single cation-π interaction in the folding dynamics of a naturally occurring α-helix peptide is uniquely revealed by these data.
对单 α-螺旋快速折叠动力学的理解主要来自于对显示出高螺旋倾向序列的合理设计肽的研究。天然存在的肽中 α-螺旋构象的折叠/去折叠动力学和热力学仍在很大程度上未被探索。在这里，我们报告了牛胰腺核糖核酸酶 A 的 C 肽类似物的蛋白质片段 RN80 的研究，RN80 是一个 13 个氨基酸残基的肽，在水溶液中采用高度流行的螺旋构象。RN80 的 H NMR 和 CD 结构研究表明，α-螺旋形成显示出 pH 依赖性的钟形曲线，在 pH 5 附近达到最大值，在碱性 pH 下，螺旋含量大幅下降。稳定这种短 α-螺旋的主要力被确定为 Glu-2 和 Arg-10 之间形成的盐桥以及涉及 Tyr-8 和 His-12 的阳离子-π 相互作用。因此，Glu-2 的去质子化或 His-12 的质子化对于 RN80 α-螺旋的稳定性是必不可少的。在本研究中，通过激光诱导的 pH 跃变触发 RN80 的折叠和展开，并通过时间分辨光声量热法 (PAC) 进行检测。光酸质子释放、氨基酸残基质子化和展开/折叠事件发生在不同的时间尺度上，并使用时间分辨 PAC 清楚地区分。由于 Glu-2 的质子化和随后破坏 Glu-2 和 Arg-10 之间稳定的盐桥，RN80 α-螺旋的部分展开以亚微秒时间范围内的浓度独立体积膨胀为特征 (0.8 mL mol，369 ns)。这种小的体积膨胀报告了在破坏暴露于溶剂的盐桥时肽骨架再水合的成本，以及骨架固有膨胀。另一方面，RN80 α-螺旋的折叠由 His-12 的质子化和随后形成阳离子-π 相互作用触发，导致微秒时间收缩 (-6.0 mL mol，∼1.7 μs)。这些数据独特地揭示了两个离散侧链相互作用，即盐桥，特别是单个阳离子-π 相互作用在天然存在的 α-螺旋肽折叠动力学中的重要作用。
