Fluorescence correlation spectroscopy studies of peptide and protein binding to phospholipid vesicles
Fluorescence correlation spectroscopy studies of peptide and protein binding to phospholipid vesicles
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DOI：
10.1529/biophysj.104.039958
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发表时间：
2004-08-01
影响因子：
3.4
通讯作者：
Rädler, J
中科院分区：
文献类型：
--
作者：
Rusu, L;Gambhir, A;Rädler, J
我们使用荧光相关光谱（FCS）来分析荧光标记的肽与脂质囊泡的结合，并将推导出的结合常数与使用其他技术获得的结合常数进行比较。我们使用了一种充分表征的肽，其对应于肉豆蔻酰化的富含丙氨酸的C激酶底物MARCKS（151-175）的基本效应结构域，该肽用Alexa 488荧光标记，并测量了其与由磷脂酰胆碱和磷脂酰丝氨酸或磷脂酰肌醇4，5-二磷酸组成的大单层囊泡（直径; 100 nm）的结合。因为大的单层囊泡明显大于肽，所以可以使用单色自相关测量来区分游离肽和结合肽的相关时间。从FCS测量计算的摩尔分配系数与使用离心技术从放射性标记的MARCKS（151-175）的结合测量获得的摩尔分配系数相当。此外，FCS可以测量以非常低的浓度（1-10纳摩尔）存在的肽的结合，这对于大多数其他技术是困难或不可能的。我们的数据表明，FCS可以是一个准确的和有价值的工具，用于研究肽和蛋白质与脂质膜的相互作用。
We used fluorescence correlation spectroscopy (FCS) to analyze the binding of fluorescently labeled peptides to lipid vesicles and compared the deduced binding constants to those obtained using other techniques. We used a well-characterized peptide corresponding to the basic effector domain of myristoylated alanine-rich C kinase substrate, MARCKS(151-175), that was fluorescently labeled with Alexa488, and measured its binding to large unilamellar vesicles (diameter; 100 nm) composed of phosphatidylcholine and phosphatidylserine or phosphatidylinositol 4,5-bisphosphate. Because the large unilamellar vesicles are significantly larger than the peptide, the correlation times for the free and bound peptide could be distinguished using single color autocorrelation measurements. The molar partition coefficients calculated from the FCS measurements were comparable to those obtained from binding measurements of radioactively labeled MARCKS(151-175) using a centrifugation technique. Moreover, FCS can measure binding of peptides present at very low concentrations (1-10 nmolar), which is difficult or impossible with most other techniques. Our data indicate FCS can be an accurate and valuable tool for studying the interaction of peptides and proteins with lipid membranes.