Driving forces of membrane protein assembly in membranes

膜蛋白在膜中组装的驱动力

• 批准号: 10698053
• 负责人: Janice L Robertson
• 金额: $ 34.8万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10698053
• 关键词: Amino Acid Sequence; Binding; Biological Models; CLC Gene; Cell membrane; Cell physiology; Cells; Computer Models; Computing Methodologies; Defect; Development; Dimerization; Dissociation; Entropy; Environment; Equilibrium; Exhibits; Fatty Acids; Fluorescence; Fluorobenzenes; Foundations; Free Energy; General anesthetic drugs; Grain; Hydrophobicity; Kinetics; Laboratories; Link; Lipid Bilayers; Lipids; Macromolecular Complexes; Measurement; Measures; Membrane; Membrane Proteins; Methods; Microscopy; Modeling; Molecular; Mutation; Nutrient; Phase; Physiological; Physiology; Property; Protein-Folding Disease; Proteins; Reaction; Regulation; Research Design; Role; Route; Sampling; Solvents; Specificity; Sterols; Structure; Temperature; Theoretical model; Thermodynamics; Time; Tryptophan; alpha helix; antiporter; computer studies; dimer; driving force; enthalpy; experimental study; interfacial; membrane assembly; molecular dynamics; molecular modeling; novel; pharmacologic; physical model; protein folding; protein structure; self assembly; single molecule; theories; wasting

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.

抽象的 膜蛋白负责控制营养，废物，能源和信息的通过 并从细胞中出来。他们是生理学的关键参与者，也是药物监管的关键目标 我们对这些蛋白质为何在细胞膜中热力学稳定的原因缺乏基本的理解。 简而言之，为什么出色的蛋白质表面在大脂质双层中找到其出色的蛋白质伴侣 忠实地组装成其稳定的本地结构？为了调查这个问题，罗伯逊实验室 已经开发了一个强大而严格的模型系统，以研究基于膜的平衡蛋白关联 在CLC-EC1 CL-/H+抗胞菌器的可逆二聚化上。通过荧光的发展和 单分子显微镜方法，现在可以实验进行完整的热力学 分析脂质双层中CLC二聚反应，产生缔合的自由能（ΔG°）和游离 由于突变或不同的脂质条件引起的能量变化（ΔΔG）。最近的进步使我们得以学习 CLC平衡是温度的函数，从而使Van't Hoff分析能够剖析热力学 二聚体后，焓（ΔH°）和熵（ΔS°）的变化。此外，我们开发了用于 在膜上和实时测量CLC二聚化的平衡动力学。 有了这个基础，Robertson Lab启动了构建CLC二聚体的完整分子模型 膜。在该项目的下一阶段，我们将调查CLC亚基被驱动到的假设 由于相关状态和解离状态中的差异依赖驱动力而导致的相关性，并且 过渡状态涉及关键的解决方案/去溶解步骤。我们将沿三个不同的 目的，通过使用计算的机理中的机制中构建CLC二聚体自由能的理论模型 方法（AIM 1），通过实验将CLC序列和结构连接到二聚化 热力学和动力学的测量（AIM 2），并开发了一种实验的客人主持人方法 并从属上量化混合脂质组成对膜中蛋白质缔合的影响 （目标3）。通过这些研究，我们整合了实验和理论，使我们能够 在物理驱动力和分子机制之间建立牢固的连接。无论有效期如何 关于我们的假设，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，我们希望这些研究的结果将为该领域提供基础 建立针对膜蛋白稳定性和基于基本物理的折叠式折叠的新策略 原则。