Single-Molecule Fluorescence Imaging of Peptide Binding to Supported Lipid Bilayers
Single-Molecule Fluorescence Imaging of Peptide Binding to Supported Lipid Bilayers
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DOI：
10.1021/ac9007682
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发表时间：
2009-07-01
影响因子：
7.4
通讯作者：
Harris, Joel M.
中科院分区：
文献类型：
--
作者：
Fox, Christopher B.;Wayment, Joshua R.;Harris, Joel M.
单分子荧光成像技术已适应于肽结合脂质双分子层的定量表征。肽-膜相互作用在治疗学、诊断学、膜渗透以及理解膜结合蛋白的结构和功能方面都很重要。全内反射荧光（TIRF）成像能够通过对单个肽分子的可靠计数来确定膜结合平衡常数，从而报告其在膜中的表面密度。单个分子在膜中的停留时间也可以确定，从停留时间的直方图确定解结合速率。解结合动力学和平衡常数的组合允许肽与膜的结合率也被报道。我们应用这种方法来表征胰高血糖素样肽-1 （GLP-1）的脂膜亲和力，GLP-1是一种参与血糖控制的30残基膜活性肽。使用单分子TIRF成像，我们测量了GLP-1与支持的磷脂双分子层的关系，确定了其结合平衡常数。观察到两种解离率，表明肽的强结合状态和弱结合状态。膜结合的速率比扩散控制的慢得多，表明膜结合存在显著的动力学障碍。数据解释使用异相，表面反应模型类似于电子转移动力学在电极上。据我们所知，这些结果是使用单分子计数来量化肽脂双分子层结合平衡和动力学的第一个例子。
Single-molecule fluorescence imaging techniques have been adapted to the quantitative characterization of peptide-binding to lipid bilayers. Peptide-membrane interactions are important in therapeutics, diagnostics, and membrane permeation and for understanding of the structure and function of membrane-bound proteins. Total-internal reflection fluorescence (TIRF) imaging is capable of determining membrane-binding equilibrium constants through the reliable counting of individual peptide molecules in order to report their surface density in the membrane. The residence times of the individual molecules in the membrane can also be determined and the rates of unbinding determined from a histogram of residence times. A combination of the unbinding kinetics and the equilibrium constant allows the binding rate of a peptide to the membrane also to be reported. We apply this method to characterize the lipid membrane affinity of glucagon-like peptide-1 (GLP-1), a 30-residue membrane-active peptide that is involved in glycemic control. Using single-molecule TIRF imaging, we have measured the affiliation of GLP-1 with a supported, phospholipid bilayer kind determined its binding equilibrium constant. Two rates of dissociation were observed, suggesting strongly and weakly bound states of the peptide. The rate of membrane association was much slower than diflusion-controlled, indicating a significant kinetic barrier to membrane binding. The data were interpreted using a heterogeneous, surface-reaction model analogous to electron-transfer kinetics at an electrode. To our knowledge, these results are the first example of using single-molecule counting to quantify peptide-lipid bilayer binding equilibria and kinetics.