Dual time-resolved temperature-jump fluorescence and infrared spectroscopy for the study of fast protein dynamics.
Dual time-resolved temperature-jump fluorescence and infrared spectroscopy for the study of fast protein dynamics.
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DOI：
10.1016/j.saa.2017.01.069
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发表时间：
2017-05-05
期刊：
影响因子：
--
通讯作者：
Dyer RB
中科院分区：
文献类型：
--
作者：
Davis CM;Reddish MJ;Dyer RB
时间分辨温度跃变（T-jump）与荧光和红外（IR）光谱相结合是监测蛋白质动力学的一种强大技术。虽然多肽酰胺I模式的红外光谱在技术上更具挑战性，但它提供了补充信息，因为它直接探测蛋白质骨架的变化，而荧光光谱对特定侧链的环境敏感。随着宽调谐量子级联激光器（QCL）的出现，可以以高灵敏度和再现性有效地探测多个IR频率。本文描述了一种时间分辨T-跳跃荧光和红外光谱仪及其在蛋白质折叠动力学研究中的应用。Q开关Ho：YAG激光器为时间分辨IR和荧光光谱提供了T跳跃源，分别由QCL和Ti：Sapphire激光器探测。Ho：YAG激光器同时泵浦时间分辨IR和荧光光谱仪。仪器灵敏度高，红外吸收检测限小于0.2 mOD，荧光灵敏度为总荧光强度的2%。使用计算机控制的QCL快速调谐的IR频率，它是可能的，以创建一个T-跳跃诱导的差异光谱从50 ns到0.5 ms。这项研究表明，功率的双重时间分辨T-跳跃荧光和红外光谱，以解决复杂的折叠机制，通过互补的红外吸收和荧光测量蛋白质动力学。
Time-resolved temperature-jump (T-jump) coupled with fluorescence and infrared (IR) spectroscopy is a powerful technique for monitoring protein dynamics. Although IR spectroscopy of the polypeptide amide I mode is more technically challenging, it offers complementary information because it directly probes changes in the protein backbone, whereas, fluorescence spectroscopy is sensitive to the environment of specific side chains. With the advent of widely tunable quantum cascade lasers (QCL) it is possible to efficiently probe multiple IR frequencies with high sensitivity and reproducibility. Here we describe a dual time-resolved T-jump fluorescence and IR spectrometer and its application to study protein folding dynamics. A Q-switched Ho:YAG laser provides the T-jump source for both time-resolved IR and fluorescence spectroscopy, which are probed by a QCL and Ti:Sapphire laser, respectively. The Ho:YAG laser simultaneously pumps the time-resolved IR and fluorescence spectrometers. The instrument has high sensitivity, with an IR absorbance detection limit of less than 0.2 mOD and a fluorescence sensitivity of 2% of the overall fluorescence intensity. Using a computer controlled QCL to rapidly tune the IR frequency it is possible to create a T-jump induced difference spectrum from 50 ns to 0.5 ms. This study demonstrates the power of the dual time-resolved T-jump fluorescence and IR spectroscopy to resolve complex folding mechanisms by complementary IR absorbance and fluorescence measurements of protein dynamics.