Driving forces of membrane protein assembly in membranes

膜蛋白在膜中组装的驱动力

• 批准号: 10298719
• 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/10298719
• 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; Pharmacology; 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; antiporter; base; computer studies; dimer; driving force; enthalpy; experimental study; interfacial; membrane assembly; molecular dynamics; molecular modeling; novel; 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物质、废物、能量和信息的通过 并脱离细胞。它们是生理学的关键参与者和药理调节的关键目标，但是， 我们对为什么这些蛋白质在细胞膜中热力学稳定缺乏基本的了解。 简而言之，为什么油腻的蛋白质表面会在油腻的脂质双层中找到其油腻的蛋白质伴侣 忠实地组装成其稳定的原生结构？为了研究这个问题，罗伯逊实验室 开发了一个强大而严格的模型系统来研究基于膜的平衡蛋白质关联 关于 CLC-ec1 Cl-/H+ 反向转运蛋白的可逆二聚化。通过荧光和 单分子显微镜方法，现在可以通过实验进行完整的热力学 分析脂质双层中的 CLC 二聚反应，产生缔合自由能 (ΔG°) 和自由能 由于突变或不同的脂质条件而导致的能量变化（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实时测量 CLC 二聚化的平衡动力学。 有了这个基础，Robertson 实验室就准备建立 CLC 二聚化的完整分子模型 膜。在该项目的下一阶段，我们将研究 CLC 亚基被驱动的假设 由于缔合和解离状态下不同的溶剂依赖性驱动力而缔合，并且 过渡态涉及关键的溶剂化/去溶剂化步骤。我们将沿着三个不同的方向对此进行调查 目标是通过使用计算建立膜中 CLC 二聚自由能的理论模型 方法（目标 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），并开发宾主实验方法 并从理论上量化混合脂质成分对膜中蛋白质缔合平衡的影响 （目标 3）。在每一项研究中，我们将实验和理论紧密结合起来，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。与有效性无关 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，我们期望这些研究的结果将为该领域提供基础 基于基本物理原理建立针对膜蛋白稳定性和错误折叠的新策略 原则。