Institute of Molecular Science, Universitat de València, 46980 Paterna, Spain.
Chem Rev. 2020 Apr 8;120(7):3466-3576. doi: 10.1021/acs.chemrev.9b00449. Epub 2020 Mar 23.
Infrared difference spectroscopy probes vibrational changes of proteins upon their perturbation. Compared with other spectroscopic methods, it stands out by its sensitivity to the protonation state, H-bonding, and the conformation of different groups in proteins, including the peptide backbone, amino acid side chains, internal water molecules, or cofactors. In particular, the detection of protonation and H-bonding changes in a time-resolved manner, not easily obtained by other techniques, is one of the most successful applications of IR difference spectroscopy. The present review deals with the use of perturbations designed to specifically change the protein between two (or more) functionally relevant states, a strategy often referred to as reaction-induced IR difference spectroscopy. In the first half of this contribution, I review the technique of reaction-induced IR difference spectroscopy of proteins, with special emphasis given to the preparation of suitable samples and their characterization, strategies for the perturbation of proteins, and methodologies for time-resolved measurements (from nanoseconds to minutes). The second half of this contribution focuses on the spectral interpretation. It starts by reviewing how changes in H-bonding, medium polarity, and vibrational coupling affect vibrational frequencies, intensities, and bandwidths. It is followed by band assignments, a crucial aspect mostly performed with the help of isotopic labeling and site-directed mutagenesis, and complemented by integration and interpretation of the results in the context of the studied protein, an aspect increasingly supported by spectral calculations. Selected examples from the literature, predominately but not exclusively from retinal proteins, are used to illustrate the topics covered in this review.
红外差谱技术探测蛋白质在受到扰动时的振动变化。与其他光谱方法相比，它在探测蛋白质中不同基团的质子化状态、氢键和构象方面具有独特的优势，这些基团包括肽骨架、氨基酸侧链、内部水分子或辅因子。特别是，以时间分辨的方式检测质子化和氢键的变化，这是红外差谱技术最成功的应用之一，而这些变化往往难以通过其他技术获得。本综述涉及使用设计用于特定改变蛋白质在两个（或更多）功能相关状态之间的扰动，这一策略通常被称为反应诱导的红外差谱技术。在本综述的前半部分，我回顾了蛋白质的反应诱导红外差谱技术，特别强调了合适样品的制备及其特性、蛋白质的扰动策略以及时间分辨测量的方法学（从纳秒到分钟）。本综述的后半部分重点介绍光谱解释。它首先回顾了氢键、介质极性和振动耦合的变化如何影响振动频率、强度和带宽。然后进行了带分配，这是一个关键的方面，主要通过同位素标记和定点突变来完成，并通过在研究蛋白质的背景下整合和解释结果来补充，这一方面越来越受到光谱计算的支持。本文选择了一些来自文献的例子，主要但不限于视网膜蛋白的例子，来说明本综述涵盖的主题。
