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

基本信息

批准号：
8721977
负责人：
Janice L Robertson
金额：
$ 24.38万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
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/8721977
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

项目摘要

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对于可溶性蛋白质，疏水基团被埋藏。远离水界面是主要驱动力，但膜嵌入蛋白质无法经历由于脂质双层缺乏水，因此出现了疏水力：一个基本难题是如何产生的。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+ 的结构稳定且功能性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。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人口进行量化：(i) ¿泊松计数¿单体对比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¿斯特尔共振通过这些测定，可以在脂质体和支持的双层中进行能量转移（FRET）。到位后，将进行实验来研究已经普遍存在的两种替代假设首先，特定的跨膜螺旋相互作用是由范德驱动的。高度互补表面的瓦尔斯力的变化将在替换时测量。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是。由螺旋缔合时脂质的熵增加驱动，为了研究这一点，形成脂质的分子。通过改变化学头基、链长和链序来修改溶剂对于所有实验，自由能关系也将是来自古细菌的不饱和或四醚脂质。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。