Driving forces of membrane protein assembly in membranes

摘要 膜蛋白负责控制营养物质、废物、能量和信息的传递。 和细胞外。然而，它们是生理学上的关键参与者，也是药物调节的关键目标， 我们缺乏对为什么这些蛋白质在细胞膜中热力学稳定的基本理解。 简单地说，为什么一个油腻的蛋白质表面在油腻的脂类双层中找到了它的油腻蛋白质伙伴 忠实地组装成它稳定的、原生的结构？为了研究这个问题，罗伯逊实验室 已经开发了一个健壮而严谨的模型系统来研究膜中平衡的蛋白质结合。 ClC-EC1Cl-/H逆向转运蛋白的可逆二聚反应。通过荧光和荧光的发展 单分子显微镜的出现，现在可以通过实验进行完整的热力学 类脂双层中ClC二聚反应的分析，产生自由缔合能(ΔG°)和自由 能量变化(ΔΔG)由突变或不同的脂质条件引起。最近的进展使我们能够研究 CLC平衡作为温度的函数，使得范特霍夫分析能够剖析热力学 二聚反应时的焓(ΔH°)和熵(ΔS°)的变化。此外，我们还开发了一些方法来 以一种轻松的方式测量CLC二聚的平衡动力学--在膜中并实时测量。 有了这个基础，罗伯逊实验室就可以建立一个完整的CLC二聚化分子模型 膜。在这个项目的下一阶段，我们将研究CLC亚基被驱动到 由于在缔合和解离状态下依赖于不同溶剂的驱动力而缔合，并且 过渡态包括一个关键的溶剂化/去溶剂化步骤。我们将从三个不同的角度对此进行调查 旨在通过计算建立膜中ClC二聚自由能的理论模型 途径(目标1)，通过实验将ClC序列和结构与二聚化联系起来 热力学和动力学的测量(目标2)，并开发一种宾主方法来进行实验 并从理论上量化了混合脂组成对膜中蛋白质结合平衡的影响 (目标3)。在每一项研究中，我们都将实验和理论紧密结合在一起，使我们能够 在物理驱动力和分子机制之间建立牢固的联系。不管有效性如何 根据我们的假设，我们的研究将为膜蛋白的研究提供有意义的定量信息 在膜中。最终，我们预计这些研究的结果将为实地提供一个基础，以 基于基础物理构建靶向膜蛋白稳定性和错误折叠的新策略 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.