Chung Hoi Sung, Tokmakoff Andrei
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Proteins. 2008 Jul;72(1):474-87. doi: 10.1002/prot.22043.
Transient thermal unfolding of ubiquitin is investigated using nonlinear infrared spectroscopy after a nanosecond laser temperature jump (T-jump). The abrupt change in the unfolding free energy surface and the ns time resolution allow us to observe a fast response on ns to micros time-scales, which we attribute to downhill unfolding, before a cross-over to ms kinetics. The downhill unfolding by a sub-population of folded proteins is induced through a shift of the barrier toward the native state. By adjusting the T-jump width, the effect of the initial (T(i)) and final (T(f)) temperature on the unfolding dynamics can be separated. From the amplitude of the fast downhill unfolding, the fractional population prepared at the unfolding transition state is obtained. This population increases with both T(i) and with T(f). A two-state kinetic analysis of the ms refolding provides thermodynamic information about the barrier height. By a combination of the fast and slow unfolding and folding parameters, a quasi-two-state kinetic analysis is performed to calculate the time-dependent population changes of the folded state. This calculation coincides with the experimentally obtained population changes at low temperature but deviations are found in the T-jump from 67 to 78 degrees C. Using temperature-dependent barrier height changes, a temperature Phi value analysis is performed. The result shows a decreasing trend of Phi(T) with temperature, which indicates an increase of the heterogeneity of the transition state. We conclude that ubiquitin unfolds along a well-defined pathway at low temperature which expands with increasing temperature to include multiple routes.
利用纳秒激光温度跃升（T-jump）后的非线性红外光谱研究了泛素的瞬态热去折叠。去折叠自由能表面的突然变化以及纳秒级的时间分辨率使我们能够观察到在纳秒到微秒时间尺度上的快速响应，我们将其归因于下坡去折叠，这发生在转变为毫秒级动力学之前。折叠蛋白亚群的下坡去折叠是通过势垒向天然态的移动诱导的。通过调整T-jump宽度，可以分离初始温度（T(i)）和最终温度（T(f)）对去折叠动力学的影响。从快速下坡去折叠的幅度，可以得到在去折叠过渡态制备的分数群体。该群体随T(i)和T(f)均增加。对毫秒级重折叠的双态动力学分析提供了有关势垒高度的热力学信息。通过结合快速和慢速去折叠及折叠参数，进行了准双态动力学分析以计算折叠态随时间的群体变化。该计算在低温下与实验获得的群体变化一致，但在67至78摄氏度的T-jump中发现了偏差。利用与温度相关的势垒高度变化，进行了温度Phi值分析。结果表明Phi(T)随温度呈下降趋势，这表明过渡态的异质性增加。我们得出结论，泛素在低温下沿明确的途径去折叠，该途径随温度升高而扩展以包括多种途径。
