Driving forces of membrane protein assembly in membranes

摘要 膜蛋白负责控制营养物质，废物，能量和信息的通过， 走出细胞。然而，它们是生理学中的关键参与者和药理学调节的关键靶点， 我们对为什么这些蛋白质在细胞膜中是化学稳定的缺乏基本的理解。 简单地说，为什么油腻的蛋白质表面会在油腻的脂质双层中找到它的油腻的蛋白质伴侣， 忠实地组装成稳定的原生结构为了研究这个问题，罗伯逊实验室 已经开发了一个强大而严格的模型系统，以研究膜中的平衡蛋白质缔合， 对CLC-ec 1 Cl-/H+反向转运蛋白可逆二聚化的影响。通过荧光的发展， 单分子显微镜的方法，现在有可能在实验上进行一个完整的热力学 分析脂质双层中的CLC二聚化反应，产生缔合自由能（ΔG°）和自由能（ΔG°）。 由于突变或不同的脂质条件导致的能量变化（ΔΔG）。最近的进展使我们能够研究 CLC平衡作为温度的函数，使范特霍夫分析能够剖析热力学 二聚时的焓（ΔH°）和熵（ΔS°）的变化。此外，我们还开发了方法， 以易于处理的方式-在膜中和实时地测量CLC二聚化的平衡动力学。 有了这个基础，罗伯逊实验室准备建立一个完整的CLC二聚化的分子模型， 膜。在这个项目的下一阶段，我们将研究CLC亚基被驱动的假设， 由于在缔合和解离状态下的不同溶剂依赖性驱动力而缔合，并且 过渡态包括临界溶剂化/去溶剂化步骤。我们将沿着沿着三个不同的 目的，通过建立一个理论模型的CLC二聚自由能在膜使用计算 方法（目的1），通过实验将CLC序列和结构与二聚化连接起来， 热力学和动力学测量（目标2），并开发一种宾主方法， 并从理论上量化混合脂质组合物对膜中蛋白质缔合平衡的影响 (Aim 3）。在每一项研究中，我们都将实验和理论结合起来，使我们能够 在物理驱动力和分子机制之间建立了牢固的联系。不管有效性如何 我们的研究为膜蛋白的研究提供了有意义的定量信息 在细胞膜中。最终，我们希望这些研究的结果将为该领域提供基础， 建立新的战略，针对膜蛋白的稳定性和错误折叠的基础上，基本的物理 原则

ABSTRACT Membrane proteins are responsible for controlling the passage of nutrients, waste, energy and information in and out of cells. They are critical players in physiology and key targets for pharmacological regulation, however, we lack a fundamental understanding of why these proteins are thermodynamically stable in cell membranes. Simply put, why does a greasy protein surface find its greasy protein partner in the greasy lipid bilayer to assemble faithfully into its stable, native structure? In order to investigate this question, the Robertson laboratory has developed a robust and rigorous model system to study equilibrium protein association in membranes based on the reversible dimerization of the CLC-ec1 Cl-/H+ antiporter. Through the development of fluorescence and single-molecule microscopy approaches, it is now possible to experimentally conduct a full thermodynamic analysis of the CLC dimerization reaction in lipid bilayers, yielding the free energy of association (ΔG°) and free energy changes (ΔΔG) due to mutations or different lipid conditions. Recent advances have allowed us to study CLC equilibrium as a function of temperature, enabling a van't Hoff analysis to dissect the thermodynamic changes in enthalpy (ΔH°) and entropy (ΔS°) upon dimerization. In addition, we have developed methods for measuring equilibrium kinetics of CLC dimerization in a tractable manner - in the membrane and in real-time. With this foundation in place, the Robertson lab is primed to build a full molecular model of CLC dimerization in membranes. In the next phase of this project, we will investigate the hypothesis that CLC subunits are driven to associate due to differential solvent dependent driving forces in the associated and dissociated states, and that the transition state involves a critical solvation/de-solvation step. We will investigate this along three distinct aims, by building a theoretical model of the CLC dimerization free energy in membranes using computational approaches (Aim 1), connecting the CLC sequence and structure to dimerization through experimental measurements of thermodynamics and kinetics (Aim 2), and developing a guest-host approach to experimentally and theoretically quantify the impact of mixed lipid composition on protein association equilibria in membranes (Aim 3). Throughout each of these studies, we integrate experiments and theory hand-in-hand, enabling us to make robust connections between physical driving forces and molecular mechanisms. Regardless of the validity of our hypothesis, our studies will provide meaningful quantitative information to the study of membrane proteins in membranes. Ultimately, we expect that the results from these studies will provide a foundation for the field to build new strategies for targeting membrane protein stability and mis-folding based on fundamental physical principles.