Site-Specific 1D and 2D IR Spectroscopy to Characterize the Conformations and Dynamics of Protein Molecular Recognition
Site-Specific 1D and 2D IR Spectroscopy to Characterize the Conformations and Dynamics of Protein Molecular Recognition
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位点特异性一维和二维红外光谱表征蛋白质分子识别的构象和动力学
DOI：
10.1021/acs.jpcb.9b00969
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Thielges, Megan C.
中科院分区：
文献类型：
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作者：
Ramos, Sashary;Thielges, Megan C.
蛋白质作为相互转换状态的集合体存在于复杂的能量景观中。一个完整的，分子水平的了解他们的功能需要知识的人口状态，从而实验工具来表征他们。红外（IR）光谱具有固有的快速时间尺度，可以捕获所有状态及其动力学，原则上具有键特定的空间分辨率，并且提供更丰富信息的2D IR方法正变得越来越常规。虽然红外光谱法在蛋白质研究中的应用受到光谱拥挤的挑战，但可以通过位点特异性引入具有频率分辨反射的红外探针基团的氨基酸侧链来克服这个问题，这进一步使得能够选择性地表征蛋白质中的不同位置。本文简要介绍了生物物理学方法，并对蛋白质研究的最新进展进行了综述。然后，我们描述了我们的努力，应用特定位点的一维和二维红外光谱对蛋白质的构象和动力学的阐明，调查他们参与蛋白质分子识别，特别是介导的动态复合物：质体蓝素及其结合伴侣细胞色素f，细胞色素P450和底物或氧化还原伙伴，Src同源3域和脯氨酸丰富的肽基序。我们强调了频率分辨探针的优势，以表征蛋白质中的特定局部位点，并揭示不同位置之间的变化，以及红外光谱的快速时标检测快速相互转换状态的优势。此外，我们说明了更大的洞察力所提供的二维方法，并讨论了潜在的路线，进一步推进生物分子二维红外光谱领域。
Proteins exist as ensembles of interconverting states on a complex energy landscape. A complete, molecular-level understanding of their function requires knowledge of the populated states and thus the experimental tools to characterize them. Infrared (IR) spectroscopy has an inherently fast time scale that can capture all states and their dynamics with, in principle, bond-specific spatial resolution, and 2D IR methods that provide richer information are becoming more routine. Although application of IR spectroscopy for investigation of proteins is challenged by spectral congestion, the issue can be overcome by site-specific introduction of amino acid side chains that have IR probe groups with frequency-resolved absorptions, which furthermore enables selective characterization of different locations in proteins. Here, we briefly introduce the biophysical methods and summarize the current progress toward the study of proteins. We then describe our efforts to apply site-specific 1D and 2D IR spectroscopy toward elucidation of protein conformations and dynamics to investigate their involvement in protein molecular recognition, in particular mediated by dynamic complexes: plastocyanin and its binding partner cytochromef, cytochrome P450s and substrates or redox partners, and Src homology 3 domains and proline-rich peptide motifs. We highlight the advantages of frequency-resolved probes to characterize specific, local sites in proteins and uncover variation among different locations, as well as the advantage of the fast time scale of IR spectroscopy to detect rapidly interconverting states. In addition, we illustrate the greater insight provided by 2D methods and discuss potential routes for further advancement of the field of biomolecular 2D IR spectroscopy.