Measuring Protein Binding to Lipid Vesicles by Fluorescence Cross-Correlation Spectroscopy
Measuring Protein Binding to Lipid Vesicles by Fluorescence Cross-Correlation Spectroscopy
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DOI：
10.1016/j.bpj.2017.06.023
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发表时间：
2017-09-19
影响因子：
3.4
通讯作者：
Bacia, Kirsten
中科院分区：
文献类型：
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作者：
Krueger, Daniela;Ebenhan, Jan;Bacia, Kirsten
荧光相关光谱以前已被用于研究肽和蛋白质与脂质膜的结合，因为它允许非常少量的样品，短的测量时间和平衡结合条件。然而，仅标记结合配偶体中的一个具有某些缺点，因为它依赖于通过扩散系数的变化来识别结合事件。由于肽和蛋白质聚集可以掩盖特异性结合，并且由于非化学计量结合需要明确选择结合配体数量的统计分布，因此我们另外标记脂质体并进行双色荧光互相关光谱法（dcFCCS）。我们开发了一个理论框架，表明dcFCCS幅度允许计算配体结合的程度和未结合配体的浓度，从而产生与模型无关的结合曲线。由于配体的标记程度不影响测量量，因此允许混合标记的和未标记的配体，从而扩展可用蛋白质浓度和可接近的解离常数K-D的范围。需要知道总蛋白浓度，而不是标记蛋白的分数。在这项工作中，我们将我们的dcFCCS分析方案应用于Sar 1 p，这是COPII复合物的一种蛋白质，它结合“主要-次要-混合”脂质体。Langmuir等温模型得出K-D =（2.1 +/-1.1）μ M作为单位点解离常数。这里提出的dcFCCS框架对于结合相互作用的生物物理分析是高度通用的。它可以应用于许多类型的荧光标记的配体和小的扩散颗粒，包括纳米盘和含有膜蛋白受体的脂质体。
Fluorescence correlation spectroscopy has been previously used to investigate peptide and protein binding to lipid membranes, as it allows for very low amounts of sample, short measurement times and equilibrium binding conditions. Labeling only one of the binding partners, however, comes with certain drawbacks, as it relies on identifying binding events by a change in diffusion coefficient. Since peptide and protein aggregation can obscure specific binding, and since non-stoichiometric binding necessitates the explicit choice of a statistical distribution for the number of bound ligands, we additionally label the liposomes and perform dual-color fluorescence cross-correlation spectroscopy (dcFCCS). We develop a theoretical framework showing that dcFCCS amplitudes allow calculation of the degree of ligand binding and the concentration of unbound ligand, leading to a model-independent binding curve. As the degree of labeling of the ligands does not factor into the measured quantities, it is permissible to mix labeled and unlabeled ligand, thereby extending the range of usable protein concentrations and accessible dissociation constants, K-D. The total protein concentration, but not the fraction of labeled protein, needs to be known. In this work, we apply our dcFCCS analysis scheme to Sar1p, a protein of the COPII complex, which binds "major-minor-mix'' liposomes. A Langmuir isotherm model yields K-D = (2.1 +/- 1.1) mu M as the single-site dissociation constant. The dcFCCS framework presented here is highly versatile for biophysical analysis of binding interactions. It may be applied to many types of fluorescently labeled ligands and small diffusing particles, including nanodiscs and liposomes containing membrane protein receptors.