Folding Dynamics of the Trp-Cage Miniprotein: Evidence for a Native-Like Intermediate from Combined Time-Resolved Vibrational Spectroscopy and Molecular Dynamics Simulations
Folding Dynamics of the Trp-Cage Miniprotein: Evidence for a Native-Like Intermediate from Combined Time-Resolved Vibrational Spectroscopy and Molecular Dynamics Simulations
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DOI：
10.1021/jp404714c
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发表时间：
2013-10-03
影响因子：
3.3
通讯作者：
Woutersen, Sander
中科院分区：
文献类型：
--
作者：
Meuzelaar, Heleen;Marino, Kristen A.;Woutersen, Sander
Trp-CAGE是一种人工合成的20个残基的微型蛋白，它能迅速自发地折叠成一个明确的球状结构，更典型的是较大的蛋白质。由于其体积小，折叠速度快，是研究蛋白质折叠机制的理想模型系统。然而，Trp-Cage的确切折叠机制仍然是一个有争议的问题。在这里，我们用时间分辨红外光谱研究了Trp-Cage在酰胺I光谱区(1530-1700 cm(-1))的弛豫动力学。通过将同位素标记(C-13=O-18)结合到残基Gly11的酰胺羰基中，从而光谱分离单个3(10)螺旋残基，获得残基特异性信息。通过纳秒温度跳跃(T跳跃)扰动折叠展开平衡，并通过观察酰胺I‘区随时间变化的振动响应来探索随后的重新平衡。我们在322K观察到时间常数分别为100+/-10和770+/-40 ns的双峰松弛动力学，表明折叠过程包含一个中间态，其性质可以从时间和频率分辨数据中确定。我们发现，在接近熔化温度时，松弛动力学涉及多聚脯氨酸II区的快速波动，而较慢的过程可归因于蛋白质的整体(未)折叠跃迁导致的构象重排。结合我们的T-JUMP数据和分子动力学模拟结果表明，在疏水笼状结构的快速形成之前，一个定义明确的α-螺旋的形成先于疏水笼状结构的快速形成，这意味着一个天然的类似折叠的中间体，这与C-末端多聚脯氨酸II螺旋相对于骨架的N-端部分的折叠构象主要不同。我们发现主要的自由能垒位于折叠中间体和未折叠状态系综之间，它涉及到α-螺旋、3(10)-螺旋和Asp9-Arg16盐桥的形成。我们的结果表明，在低温下(T
Trp-cage is a synthetic 20-residue miniprotein which folds rapidly and spontaneously to a well-defined globular structure more typical of larger proteins. Due to its small size and fast folding, it is an ideal model system for experimental and theoretical investigations of protein folding mechanisms. However, Trp-cage's exact folding mechanism is still a matter of debate.,Here we investigate Trp-cage's relaxation dynamics in the amide I' spectral region (1530- 1700 cm(-1)) using time-resolved infrared spectroscopy. Residue-specific information was obtained by incorporating an isotopic label (C-13=O-18) into the amide carbonyl group of residue Gly11, thereby spectrally isolating an individual 3(10)-helical residue. The folding unfolding equilibrium is perturbed using a nanosecond temperature jump (T jump), and the subsequent re-equilibration is probed by observing the time dependent vibrational response in the amide I' region. We observe bimodal relaxation kinetics with time constants of 100 +/- 10 and 770 +/- 40 ns at 322 K, suggesting that the folding involves an intermediate state, the character of which can be determined from the time and frequency resolved data We find that the relaxation dynamics close to the melting temperature involve fast fluctuations in the polyproline II region, whereas the slower process can be attributed to conformational rearrangements due to the global (un)folding transition of the protein. Combined analysis of our T-jump data and molecular dynamics simulations indicates that the formation of a well-defined alpha-helix precedes the rapid formation of the hydrophobic cage structure, implying a native like folding intermediate, that Mainly differs from the folded conformation in the orientation of the C-terminal polyproline II helix relative to the N-terminal part of the backbone., We find that the main free energy barrier is positioned between the folding intermediate and the unfolded state ensemble, and that it involves the formation of the alpha-helix, the 3(10)-helix, and the Asp9- Arg16 salt bridge. Our results suggest that at low temperature (T