Reversible dimerization of a CLC transporter: A model for membrane protein foldin

CLC 转运蛋白的可逆二聚化：膜蛋白折叠模型

• 批准号: 8714314
• 负责人: Janice L Robertson
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8714314
• 关键词: Affinity; Alanine; Amino Acids; Archaea; Back; Biochemical; Biocompatible Materials; Biological Assay; Biological Models; Burial; CLC Gene; Cell physiology; Chemicals; Crystallography; Databases; Dependency; Detergents; Dimerization; Energy Transfer; Entropy; Environment; Ethers; Excision; Fluorescence; Foundations; Free Energy; Freedom; Head; Integral Membrane Protein; Ion Transport; Lead; Length; Lipid Bilayers; Lipid Binding; Lipids; Liposomes; Maps; Measurement; Measures; Membrane; Membrane Lipids; Membrane Proteins; Methods; Micelles; Modeling; Molecular; Mutation; Nature; Phospholipids; Physiological Processes; Population; Positioning Attribute; Process; Property; Proteins; Quality Control; Scanning; Side; Signal Transduction; Solvents; Specificity; Structure; Study models; Surface; System; Temperature; Testing; Thermodynamics; Translating; Tryptophan; Water; Work; abstracting; alpha helix; antiporter; aqueous; design; dimer; driving force; enthalpy; experience; insight; monomer; protein folding; protein function; protein protein interaction; research study; scaffold; single molecule; therapeutic target; van der Waals force

Abstract The central enigma of protein folding lies in how the physical forces of nature drive a simple string of amino acids into a stable, conformationally defined protein. For soluble proteins, the burial of hydrophobic groups away from aqueous interfaces is a major driving force, but membrane-embedded proteins cannot experience hydrophobic forces, as the lipid bilayer lacks water. A fundamental conundrum thus arises: how does a greasy protein surface find its greasy protein partner in the greasy lipid bilayer to fold faithfully into its native structure? Recently, a structurally stable and functional monomeric form of the normally homodimeric Cl-/H+ antiporter CLC-ec1 was designed by introducing tryptophan mutations at the dimer interface. Preliminary studies show that the protein can be shifted back to the dimer state with additional mutations or in certain lipid conditions. These results present CLC-ec1 as a model for the study of reversible dimerization, which simplifies the protein folding process while still encompassing all of the thermodynamic properties of protein interactions in the membrane environment. To make these energetic measurements, the monomer/dimer populations will be quantified using three well-established methods: (i) ¿Poisson-counting¿ of monomer vs. dimers in liposome populations, (ii) fluorescence self-quenching in liposomes, and (iii) F¿rster resonance energy transfer (FRET) in liposomes and supported bilayers for single molecule studies. With these assays in place, experiments will be carried out to investigate two alternative hypotheses that have pervaded discourse in this field. First, that specific transmembrane helix interactions are enthalpy-driven by van der Waals forces at highly complementary surfaces. Changes in free energy will be measured upon substitution of interface residues to alanine or tryptophan, with significant positions studied further by increasing side- chain volume to modulate the van der Waals interactions. The second hypothesis is that interactions are driven by increased entropy of lipids upon helix association. To study this, the molecules forming the lipid solvent will be modified by changing the chemical head group, chain length and chain order using unsaturated or tetra-ether lipids from archaea. For all experiments, free energy relationships will also be measured with respect to temperature to extrapolate values for enthalpy and entropy. These results will provide insight into the driving forces for membrane protein interactions, and may even provide a foundation for attacking general questions underlying protein folding in the strange solvent that is the lipid bilayer.

抽象的 蛋白质折叠的核心谜团在于自然的物理力如何驱动一串简单的氨基酸 酸转化为稳定的、构象确定的蛋白质。对于可溶性蛋白质，疏水基团的埋藏 远离水界面是主要驱动力，但膜嵌入蛋白质无法经历 疏水力，因为脂质双层缺乏水。因此出现了一个基本难题：如何 油性蛋白质表面在油性脂质双层中找到其油性蛋白质伴侣，忠实地折叠成其天然结构 结构？最近，通常同型二聚体 Cl-/H+ 的结构稳定且具有功能性的单体形式 反向转运蛋白 CLC-ec1 是通过在二聚体界面引入色氨酸突变而设计的。初步的 研究表明，通过额外的突变或在某些脂质中，蛋白质可以转变回二聚体状态 状况。这些结果将 CLC-ec1 作为研究可逆二聚化的模型， 简化了蛋白质折叠过程，同时仍然包含蛋白质的所有热力学特性 膜环境中的相互作用。为了进行这些能量测量，单体/二聚体 将使用三种行之有效的方法对种群进行量化：（i）单体与单体的“泊松计数”。 脂质体群体中的二聚体，(ii) 脂质体中的荧光自猝灭，以及 (iii) F¿rster 共振 用于单分子研究的脂质体和支持双层中的能量转移（FRET）。通过这些检测 到位后，将进行实验来研究已经普遍存在的两种替代假设 该领域的话语。首先，特定的跨膜螺旋相互作用是由范德驱动的 高度互补的表面上的瓦尔斯力。自由能的变化将在替代时测量 丙氨酸或色氨酸的界面残基，通过增加侧链进一步研究重要位置 链体积来调节范德华相互作用。第二个假设是相互作用 由螺旋缔合时脂质熵增加驱动。为了研究这一点，形成脂质的分子 溶剂将通过改变化学头基、链长和链序进行改性 来自古细菌的不饱和或四醚脂质。对于所有实验，自由能关系也将是 测量温度以推断出焓和熵的值。这些结果将 深入了解膜蛋白相互作用的驱动力，甚至可以提供基础 解决了脂质双层这一奇怪溶剂中蛋白质折叠的一般问题。