Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo namesti 2, 16610 Prague 6, Czech Republic.
European Molecular Biology Laboratory (EMBL), Cell Biology & Biophysics Unit, Meyerhofstraße 1, 69117 Heidelberg, Germany.
Biochim Biophys Acta Biomembr. 2017 May;1859(5):852-859. doi: 10.1016/j.bbamem.2017.01.021. Epub 2017 Jan 22.
Solvatochromic probes are suitable tools for quantitative characterization of protein-membrane interactions. Based on diverse fluorophores these probes have different fluorescent properties and therefore demonstrate different responses when applied for sensing the interactions of biomolecules. Surprisingly, to the best of our knowledge, no systematic comparison of the sensitivities of solvatochromic dyes for monitoring protein-membrane interactions was described. Hence, a rational choice of an optimal environmentally sensitive probe for such experiments is usually not a straightforward task. In this work we developed a series of thiol-reactive fluorescent probes based on the fluorophores with high sensitivity to their environment and compared them with two widely used DNS and DMN probes. We investigated the responses of these probes to the interaction of probe-labeled presynaptic protein α-synuclein with lipid membranes. We observed that newly synthesized probes based on fluorene and chromone dyes, which combine the strongest brightness and significant changes of fluorescence intensity, demonstrated the highest sensitivity to interaction of α-synuclein with lipid membranes. They are especially beneficial for sensing in scattering media such as solutions of lipid vesicles. We show that the described probes permit quantitative measurements of α-synuclein binding to lipid membranes at low nanomolar concentrations. We developed a detailed protocol for measuring K and binding stoichiometry for interaction of soluble peripheral proteins with membranes based on the response of the environmentally sensitive fluorescent probes. We applied this protocol for quantification of the affinity of α-synuclein to anionic membranes and found that it is substantially higher than it was earlier reported.
溶剂探针是定量研究蛋白质-膜相互作用的一种合适工具。这些探针基于不同的荧光团，具有不同的荧光特性，因此在用于检测生物分子相互作用时会表现出不同的响应。令人惊讶的是，据我们所知，目前还没有系统比较溶剂探针监测蛋白质-膜相互作用的灵敏度的研究。因此，对于此类实验，通常很难选择出最佳的环境敏感探针。在这项工作中，我们开发了一系列基于对环境高度敏感的荧光团的硫醇反应性荧光探针，并将它们与两种广泛使用的 DNS 和 DMN 探针进行了比较。我们研究了这些探针对探针标记的突触前蛋白α-突触核蛋白与脂质膜相互作用的响应。我们观察到，新合成的基于芴和色酮染料的探针，结合了最强的亮度和荧光强度的显著变化，对α-突触核蛋白与脂质膜的相互作用表现出最高的灵敏度。它们特别有利于在散射介质（如脂质囊泡溶液）中进行传感。我们表明，所描述的探针可以在低纳摩尔浓度下定量测量α-突触核蛋白与脂质膜的结合。我们开发了一种详细的协议，用于基于环境敏感荧光探针的响应来测量可溶性外周蛋白与膜的相互作用的 K 和结合化学计量比。我们应用该协议来量化α-突触核蛋白与阴离子膜的亲和力，发现其亲和力远高于之前报道的亲和力。
