Department of Chemistry, James Franck Institute, and Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois 60637, United States.
J Phys Chem B. 2020 Oct 1;124(39):8665-8677. doi: 10.1021/acs.jpcb.0c07177. Epub 2020 Sep 21.
Pulsed temperature-jump (T-jump) spectroscopy with infrared (IR) detection has been widely used to study biophysical processes occurring from nanoseconds to ∼1 ms with structural sensitivity. However, many systems exhibit structural dynamics on time scales longer than the millisecond barrier that is set by the time scale for thermal relaxation of the sample. We developed a linear and nonlinear infrared spectrometer coupled to an intensity-modulated continuous wave (CW) laser to probe T-jump-initiated chemical reactions from <1 ms to seconds. Time-dependent modulation of the CW laser leads to a <1 ms heating time as well as a constant final temperature (±3%) for the duration of the heating time. Temperature changes of up to 75 °C in DO are demonstrated, allowing for nonequilibrium measurements inaccessible to standard pulsed optical T-jump setups. T-jump linear absorption, pump-probe, and two-dimensional IR (2D IR) spectroscopy are applied to the unfolding and refolding of ubiquitin and a model intercalated motif (i-motif) DNA sequence, and analysis of the observed signals is used to demonstrate the limits and utility of each method. Overall, the ability to probe temperature-induced chemical processes from <1 ms to many seconds with 2D IR spectroscopy provides multiple new avenues for time-dependent spectroscopy in chemistry and biophysics.
脉冲温度跳跃（T-jump）光谱学与红外（IR）检测相结合，已广泛用于研究从纳秒到约 1 毫秒的生物物理过程，具有结构敏感性。然而，许多系统表现出的结构动力学时间尺度超过了毫秒障碍，这是由样品热弛豫的时间尺度决定的。我们开发了一种线性和非线性红外光谱仪，与强度调制连续波（CW）激光耦合，以探测从<1 毫秒到秒的 T-jump 引发的化学反应。CW 激光的时变调制导致<1 毫秒的加热时间以及加热时间内的恒定最终温度（±3%）。在 DO 中证明了高达 75°C 的温度变化，这使得标准的脉冲光学 T-jump 装置无法进行非平衡测量。T-jump 线性吸收、泵浦探测和二维红外（2D IR）光谱学被应用于泛素和模型插入基序（i-motif）DNA 序列的展开和折叠，对观察到的信号进行分析，以证明每种方法的局限性和实用性。总的来说，用 2D IR 光谱学从<1 毫秒到许多秒探测温度诱导的化学过程的能力为化学和生物物理学中的时间相关光谱学提供了多个新途径。
