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Binding of Biotin to Streptavidin: A combined fluorescence correlation spectroscopy and time-resolved fluorescence study

机译：生物素与链霉亲和素的结合：荧光相关光谱和时间分辨荧光研究的结合

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The Biotin-Streptavidin complex is a widely studied system in biology and biophysics, because of its extremely strong non-covalent binding affinity. The latter is often exploited to link molecules to substrates or to one another. However, the details of the Biotin-Streptavidin binding have not been fully elucidated so far. Particularly, the role of cooperative effects in enhancing the binding affinity has not been clarified. Our long-term aim is to investigate this point by implementing two complementary approaches, fluorescence correlation spectroscopy and time-correlated single-photon counting. As both methods rely on the analysis of fluorescence signals, biotin labeled with Atto-550-dye was used. In this work, in order to get a first overview of the system, we analyzed solutions in three paradigmatic ranges of Biotin-to-Streptavidin concentration ratio. Fluorescence correlation spectroscopy measurements allowed us to extract diffusion times of free biotin and of biotin-Streptavidin complexes, and also to gain information about the dynamics of the intersystem crossing between the first excited triplet and the first excited singlet states. Time-correlated single-photon counting made it possible to derive the lifetimes of the different species in solution, as well as to deduce relevant information about the relative abundance of Streptavidin-complexed and free Biotin.
机译：生物素-链霉亲和素复合物由于其极强的非共价结合亲和力而在生物学和生物物理学中得到广泛研究。经常利用后者来将分子连接至底物或彼此。然而，迄今为止，尚未完全阐明生物素-链霉亲和素结合的细节。特别是，尚未阐明协同作用在增强结合亲和力中的作用。我们的长期目标是通过实施两种互补的方法来研究这一点，即荧光相关光谱法和与时间相关的单光子计数。由于两种方法均依赖于荧光信号的分析，因此使用了Atto-550染料标记的生物素。在这项工作中，为了获得系统的初步概览，我们分析了生物素与链霉亲和素浓度比的三个范式范围内的解决方案。荧光相关光谱法的测量使我们能够提取游离生物素和生物素-链霉亲和素复合物的扩散时间，并且还可以获得有关第一激发三重态和第一激发单重态之间的系统间交叉动力学的信息。与时间相关的单光子计数使推导溶液中不同物种的寿命成为可能，并推导了链霉亲和素复合物和游离生物素相对丰度的相关信息。

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