Site-specific 2D IR spectroscopy: a general approach for the characterization of protein dynamics with high spatial and temporal resolution
Site-specific 2D IR spectroscopy: a general approach for the characterization of protein dynamics with high spatial and temporal resolution
复制标题
位点特异性二维红外光谱：具有高空间和时间分辨率的蛋白质动力学表征的通用方法
DOI：
10.1039/c8cp06146g
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发表时间：
2019
影响因子：
3.3
通讯作者：
Thielges, Megan C.
中科院分区：
文献类型：
--
作者：
Ramos, Sashary;Horness, Rachel E.;Collins, Jessica A.;Haak, David;Thielges, Megan C.
蛋白质侧链的构象异质性和动力学对功能有贡献，但由于涉及的快速时间尺度和蛋白质结构的空间异质性所带来的实验挑战，研究如何准确地受阻。二维红外（2D IR）光谱的潜力，以前所未有的空间和时间分辨率测量构象异质性和动力学，激发了广泛的努力，开发具有频率分辨吸收的功能基团的氨基酸，作为其蛋白质微环境的探针。我们展示了该方法的全部优势，通过选择性地将探针对氰苯丙氨酸结合在Src同源3结构域的六个不同位点上，并应用二维红外光谱来特异性地表征异质性和动力学及其对同源配体结合的贡献。该方法揭示了广泛的微环境和对配体结合的不同反应，包括在形成蛋白质识别表面的三个相邻的保守芳香残基上。对所有标记蛋白进行的分子动力学模拟提供了对潜在异质性和动力学的深入了解。类似的二维红外光谱和位点选择探针的应用将允许表征其他蛋白质的异质性和动力学，异质性和动力学如何受到溶剂化和局部结构的影响，以及它们如何有助于生物功能。
The conformational heterogeneity and dynamics of protein side chains contribute to function, but investigating exactly how is hindered by experimental challenges arising from the fast timescales involved and the spatial heterogeneity of protein structures. The potential of two-dimensional infrared (2D IR) spectroscopy for measuring conformational heterogeneity and dynamics with unprecedented spatial and temporal resolution has motivated extensive effort to develop amino acids with functional groups that have frequency-resolved absorptions to serve as probes of their protein microenvironments. We demonstrate the full advantage of the approach by selective incorporation of the probe p-cyanophenylalanine at six distinct sites in a Src homology 3 domain and the application of 2D IR spectroscopy to site-specifically characterize heterogeneity and dynamics and their contribution to cognate ligand binding. The approach revealed a wide range of microenvironments and distinct responses to ligand binding, including at the three adjacent, conserved aromatic residues that form the recognition surface of the protein. Molecular dynamics simulations performed for all the labeled proteins provide insight into the underlying heterogeneity and dynamics. Similar application of 2D IR spectroscopy and site-selective probe incorporation will allow for the characterization of heterogeneity and dynamics of other proteins, how heterogeneity and dynamics are affected by solvation and local structure, and how they might contribute to biological function.