Abbyad Paul, Shi Xinghua, Childs William, McAnaney Tim B, Cohen Bruce E, Boxer Steven G
Department of Chemistry, Stanford University, Stanford, California 94305-5080, USA.
J Phys Chem B. 2007 Jul 19;111(28):8269-76. doi: 10.1021/jp0709104. Epub 2007 Jun 26.
Proteins respond to electrostatic perturbations through complex reorganizations of their charged and polar groups, as well as those of the surrounding media. These solvation responses occur both in the protein interior and on its surface, though the exact mechanisms of solvation are not well understood, in part because of limited data on the solvation responses for any given protein. Here, we characterize the solvation kinetics at sites throughout the sequence of a small globular protein, the B1 domain of streptococcal protein G (GB1), using the synthetic fluorescent amino acid Aladan. Aladan was incorporated into seven different GB1 sites, and the time-dependent Stokes shift was measured over the femtosecond to nanosecond time scales by fluorescence upconversion and time-correlated single photon counting. The seven sites range from buried within the protein core to fully solvent-exposed on the protein surface, and are located on different protein secondary structures including beta-sheets, helices, and loops. The dynamics in the protein sites were compared against the free fluorophore in buffer. All protein sites exhibited an initial, ultrafast Stokes shift on the subpicosecond time scale similar to that observed for the free fluorophore, but smaller in magnitude. As the probe is moved from the surface to more buried sites, the dynamics of the solvation response become slower, while no clear correlation between dynamics and secondary structure is observed. We suggest that restricted movements of the surrounding protein residues give rise to the observed long time dynamics and that such movements comprise a large portion of the protein's solvation response. The proper treatment of dynamic Stokes shift data when the time scale for solvation is comparable to the fluorescence lifetime is discussed.
蛋白质通过其带电和极性基团以及周围介质的复杂重组来响应静电扰动。这些溶剂化响应发生在蛋白质内部及其表面，尽管溶剂化的确切机制尚未完全理解，部分原因是关于任何给定蛋白质的溶剂化响应的数据有限。在这里，我们使用合成荧光氨基酸阿拉丹（Aladan）来表征小球蛋白链球菌蛋白G（GB1）的B1结构域整个序列位点的溶剂化动力学。阿拉丹被掺入七个不同的GB1位点，并通过荧光上转换和时间相关单光子计数在飞秒到纳秒的时间尺度上测量时间相关的斯托克斯位移。这七个位点范围从埋在蛋白质核心内到完全暴露在蛋白质表面的溶剂中，并且位于不同的蛋白质二级结构上，包括β-折叠、螺旋和环。将蛋白质位点的动力学与缓冲液中的游离荧光团进行比较。所有蛋白质位点在亚皮秒时间尺度上都表现出初始的超快斯托克斯位移，类似于游离荧光团观察到的，但幅度较小。随着探针从表面移动到更深埋的位点，溶剂化响应的动力学变得更慢，而未观察到动力学与二级结构之间的明显相关性。我们认为周围蛋白质残基的受限运动导致了观察到的长时间动力学，并且这种运动构成了蛋白质溶剂化响应的很大一部分。讨论了当溶剂化时间尺度与荧光寿命相当时动态斯托克斯位移数据的正确处理方法。
