Electrostatics Mediates the Membrane Binding of Src, a Myristoylated Protein

9419175麦克劳克林总体目标是了解含有肉豆蔻酸酯的蛋白质与生物膜相互作用的分子机制。肉豆蔻酸酯是一种14碳酰链。该建议侧重于Src(v-src癌基因的产物v-Src及其细胞同源基因c-Src，统称为Src)；工作假设是，膜结合既需要NH2末端的肉豆蔻酸盐疏水插入双层，也需要一簇相邻的碱性残基与酸性脂质的静电相互作用。具体项目有四个。1)建立了一个简单的模型，描述了肉豆蔻酸盐和碱性残基对Src与磷脂膜结合的协同作用，并通过测量与Src的NH2-末端区域相对应的肉豆蔻酸化和非肉豆蔻酸化的多肽的膜结合来检验该模型。2)通过测定天然v-Src和c-Src以及这些蛋白的突变体和嵌合体的膜结合，证实了Src NH2-末端区域的碱性残基是蛋白质-膜相互作用的静电成分。3)测定C和A蛋白的磷酸化对Src与相应多肽结合的影响。4)构建了磷脂双层和Src的NH2-末端区域的分子模型，然后用非线性Poisson-Boltzmann方程从理论上计算了结合的静电分量。该项目意义重大，因为膜结合对Src的功能至关重要。出现的生物物理原理应该适用于其他一些肉豆蔻化(例如Marcks)和法尼化(例如K-RAS)蛋白质。%总体目标是了解含有肉豆蔻酸酯的蛋白质与生物膜相互作用的分子机制。该建议侧重于Src(v-src癌基因的产物v-Src，其功能是转化m细胞，以及它的细胞同系物，c-Src，参与细胞周期的控制，统称为Src)；工作假设是，膜结合既需要NH2-末端肉豆蔻酸盐疏水插入双层，也需要一簇相邻的碱性残基与酸性脂质的静电相互作用。这是四个具体的项目。1)建立了一个简单的模型，描述了肉豆蔻酸盐和碱性残基对Src与磷脂膜结合的协同作用，并通过测量合成肽的膜结合来检验该模型。2)通过测定天然v-Src和c-Src以及这些蛋白的突变体和嵌合体的膜结合，证实了Src NH2-末端区域的碱性残基是蛋白质-膜相互作用的静电成分。3)测定C和A蛋白的磷酸化对Src与相应多肽结合的影响。4)构建了磷脂双层和Src的NH2-末端区域的分子模型，并从理论上计算了结合的静电分量。该项目意义重大，因为膜结合对Src的功能至关重要，而出现的生物物理原理应该适用于其他肉豆蔻酰化蛋白质。***

9419175 McLaughlin The overall objective is to understand the molecular mechanism by which proteins containing myristate, a 14 carbon acyl chain, interact with biological membranes. The proposal focuses on Src (the product of the v-src oncogene, v-Src, and its cellular homolog, c-Src, are known collectively as Src); the working hypothesis is that membrane binding requires both the hydrophobic insertion of the NH2-terminal myristate into the bilayer and the electrostatic interaction of a cluster of adjacent basic residues with acidic lipids. There are four specific projects. 1) Developing a simple model that describes the synergistic effect of myristate and basic residues on the binding of Src to phospholipid membranes and testing this model by measuring the membrane binding of myristoylated and nonmyristoylated peptides corresponding to the NH2-terminal region of Src. 2) Confirming that the basic residues in the NH2-terminal region of Src are responsible for the electrostatic component of protein-membrane interaction by measuring the membrane binding of native v-Src and c-Src as well as mutant and chimeric versions of these proteins. 3) Measuring the effect of phosphorylation by C and A kinases on the membrane binding of both Src and corresponding peptides. 4) Constructing a molecular model of a phospholipid bilayer and the NH2-terminal region of Src, then using the nonlinear Poisson-Boltzmann equation to calculate theoretically the electrostatic component of the binding. The project is significant because membrane binding is crucial to the function of Src. The biophysical principles that emerge should be applicable to some other myristoylated (e.g. MARCKS) and farnesylated (e.g. K-Ras) proteins. %%% The overall objective is to understand the molecular mechanism by which proteins containing myristate, a 14 carbon acyl chain, interact with biological membranes. The proposal focuses on Src (the product of the v-src oncogene, v-Src, which functions to transfor m cells, and its cellular homolog, c-Src, which is involved in control of the cell cycle, are known collectively as Src); the working hypothesis is that membrane binding requires both the hydrophobic insertion of the NH2-terminal myristate into the bilayer and the electrostatic interaction of a cluster of adjacent basic residues with acidic lipids. These are four specific projects. 1) Developing a simple model that describes the synergistic effect of myristate and basic residues on the binding of Src to phospholipid membranes and testing this model by measuring the membrane binding of synthetic peptides. 2) Confirming that the basic residues in the NH2-terminal region of Src are responsible for the electrostatic component of protein-membrane interaction by measuring the membrane binding of native v-Src and c-Src as well as mutant and chimeric versions of these proteins. 3) Measuring the effect of phosphorylation by C and A kinases on the membrane binding of both Src and corresponding peptides. 4) Constructing a molecular model of a phospholipid bilayer and the NH2-terminal region of Src, then calculating theoretically the electrostatic component of the binding. The project is significant because membrane binding is crucial to the function of Src and the biophysical principles that emerge should be applicable to other myristoylated proteins. ***