Bähr G, Diederich A, Vergères G, Winterhalter M
Department of Biophysical Chemistry, Biozentrum of the University of Basel, Switzerland.
Biochemistry. 1998 Nov 17;37(46):16252-61. doi: 10.1021/bi981765a.
We have investigated the binding of the effector domains of myristoylated alanine-rich C kinase substrate (MARCKS) and of MARCKS-related protein (MRP) to lipid model membranes. For membrane systems we used lipid monolayers on a Langmuir trough and black lipid membranes (BLM). The binding of the peptides was detected by monitoring changes in the boundary potential of the lipid membranes. The vibrating plate technique (VPT) and the method of inner field compensation (IFC) were used for the monolayer and for the BLM, respectively. We could show that the effector domain of MARCKS binds to acidic lipid membranes mainly via electrostatic interactions and to zwitterionic lipid membranes via hydrophobic interactions. Isobaric measurements on lipid monolayers revealed that binding of both effector domains is accompanied by partial insertion of the peptides into the membrane. Adsorption and insertion of the peptides could be followed simultaneously by the VPT and by recording the increase in area of the lipid monolayer, respectively. No temporal delay could be observed between adsorption and insertion of the peptides, demonstrating that adsorption is the rate-limiting step and that insertion is faster than the time resolution of the experiments, i.e., a few seconds. Both the IFC and the VPT did not show any significant difference between the behaviors of the effector domains of MARCKS and MRP. With the IFC we show that calcium can regulate the translocation of the MARCKS effector peptide between the membrane and calmodulin (CaM) in the bulk. Our results indicate, that the IFC and VPT are suitable qualitatively, and to a certain extent quantitatively, as membrane binding assays.
我们研究了肉豆蔻酰化富含丙氨酸的蛋白激酶C底物（MARCKS）和MARCKS相关蛋白（MRP）的效应结构域与脂质模型膜的结合情况。对于膜系统，我们使用了Langmuir槽上的脂质单层和黑脂质膜（BLM）。通过监测脂质膜边界电位的变化来检测肽的结合。分别使用振动板技术（VPT）和内场补偿法（IFC）来检测单层脂质膜和BLM。我们发现，MARCKS的效应结构域主要通过静电相互作用与酸性脂质膜结合，通过疏水相互作用与两性离子脂质膜结合。脂质单层的等压测量表明，两个效应结构域的结合都伴随着肽部分插入膜中。肽的吸附和插入可分别通过VPT和记录脂质单层面积的增加同时进行跟踪。在肽的吸附和插入之间未观察到时间延迟，这表明吸附是限速步骤，插入比实验的时间分辨率（即几秒钟）更快。IFC和VPT均未显示MARCKS和MRP的效应结构域行为之间存在任何显著差异。通过IFC我们表明，钙可以调节MARCKS效应肽在膜和本体中的钙调蛋白（CaM）之间的转运。我们的结果表明，IFC和VPT在定性上以及在一定程度上在定量上都适合作为膜结合测定方法。
