Department of Chemistry, The Scripps Research Institute , La Jolla, California 92037, United States.
Chem Rev. 2017 Feb 8;117(3):1927-1969. doi: 10.1021/acs.chemrev.6b00625. Epub 2017 Jan 20.
Vibrational spectroscopy provides a direct route to the physicochemical characterization of molecules. While both IR and Raman spectroscopy have been used for decades to provide detailed characterizations of small molecules, similar studies with proteins are largely precluded due to spectral congestion. However, the vibrational spectra of proteins do include a "transparent window", between ∼1800 and ∼2500 cm, and progress is now being made to develop site-specifically incorporated carbon-deuterium (C-D), cyano (CN), thiocyanate (SCN), and azide (N) "transparent window vibrational probes" that absorb within this window and report on their environment to facilitate the characterization of proteins with small molecule-like detail. This Review opens with a brief discussion of the advantages and limitations of conventional vibrational spectroscopy and then discusses the strengths and weaknesses of the different transparent window vibrational probes, methods by which they may be site-specifically incorporated into peptides and proteins, and the physicochemical properties they may be used to study, including electrostatics, stability and folding, hydrogen bonding, protonation, solvation, dynamics, and interactions with inhibitors. The use of the probes to vibrationally image proteins and other biomolecules within cells is also discussed. We then present four case studies, focused on ketosteroid isomerase, the SH3 domain, dihydrofolate reductase, and cytochrome c, where the transparent window vibrational probes have already been used to elucidate important aspects of protein structure and function. The Review concludes by highlighting the current challenges and future potential of using transparent window vibrational probes to understand the evolution and function of proteins and other biomolecules.
振动光谱为分子的物理化学特性提供了直接途径。虽然近红外和拉曼光谱已经使用了几十年，对小分子提供了详细的特性描述，但由于光谱拥挤，类似的蛋白质研究在很大程度上受到限制。然而，蛋白质的振动光谱确实包含一个“透明窗口”，在约 1800 至 2500cm 之间，现在正在开发特定位置掺入的碳-氘（C-D）、氰基（CN）、硫氰酸根（SCN）和叠氮化物（N）“透明窗口振动探针”，这些探针在该窗口内吸收，并报告其环境，以促进具有小分子样细节的蛋白质的特性描述。本综述首先简要讨论了常规振动光谱的优点和局限性，然后讨论了不同透明窗口振动探针的优缺点、将它们特异性掺入肽和蛋白质的方法，以及可以用来研究的物理化学性质，包括静电、稳定性和折叠、氢键、质子化、溶剂化、动力学以及与抑制剂的相互作用。还讨论了探针在细胞内对蛋白质和其他生物分子进行振动成像的用途。然后，我们提出了四个案例研究，重点关注酮固醇异构酶、SH3 结构域、二氢叶酸还原酶和细胞色素 c，其中透明窗口振动探针已经被用于阐明蛋白质结构和功能的重要方面。综述最后强调了使用透明窗口振动探针来理解蛋白质和其他生物分子的进化和功能的当前挑战和未来潜力。
