Conformational changes during the nanosecond-to-millisecond unfolding of ubiquitin
Conformational changes during the nanosecond-to-millisecond unfolding of ubiquitin
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DOI：
10.1073/pnas.0408646102
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发表时间：
2005-01-18
影响因子：
11.1
通讯作者：
Tokmakoff, A
中科院分区：
文献类型：
--
作者：
Chung, HS;Khalil, M;Tokmakoff, A
利用酰胺I振动的非线性红外光谱研究了泛素热去折叠过程中的稳态和瞬态构象变化。平衡温度依赖性二维红外光谱揭示了通过损失的交叉峰之间产生的离域振动的β-折叠的泛蛋白的β-折叠的展开。纳秒温度跳变后的瞬态展开与分散振动回波光谱，二维红外光谱的投影监测。而平衡研究遵循一个简单的两态展开，瞬态实验观察到复杂的弛豫行为，不同的光谱成分和跨越60年的时间。瞬态行为可以分为快和慢的时间尺度。从100纳秒到0.5毫秒，与p-片展开放松的顺序，非指数的方式，与时间常数为3 μ s和80 μ s的光谱特征。通过模拟泛素的酰胺I振动，这一观察被解释为展开的不太稳定的链III-V的β-片展开前的发夹，形成的疏水核心的一部分。这种下坡展开之后是指数跨越障碍的动力学在3毫秒的时间尺度。
Steady-state and transient conformational changes upon the thermal unfolding of ubiquitin were investigated with nonlinear IR spectroscopy of the amide I vibrations. Equilibrium temperaturedependent 2D IR spectroscopy reveals the unfolding of the beta-sheet of ubiquitin through the loss of cross peaks formed between transitions arising from delocalized vibrations of the beta-sheet. Transient unfolding after a nanosecond temperature jump is monitored with dispersed vibrational echo spectroscopy, a projection of the 2D IR spectrum. Whereas the equilibrium study follows a simple two-state unfolding, the transient experiments observe complex relaxation behavior that differs for various spectral components and spans 6 decades in time. The transient behavior can be separated into fast and slow time scales. From 100 ns to 0.5 ms, the spectral features associated with p-sheet unfolding relax in a sequential, nonexponential manner, with time constants of 3 mus and 80 mus. By modeling the amide I vibrations of ubiquitin, this observation is explained as unfolding of the less stable strands lll-V of the beta-sheet before unfolding of the hairpin that forms part of the hydrophobic core. This downhill unfolding is followed by exponential barrier-crossing kinetics on a 3-ms time scale.