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中心谜团在于自然的物理力如何驱动简单的氨基酸成稳定，构象的蛋白质。对于固体蛋白质，埋葬疏水基团远离水界面是主要的驱动力，但是膜包裹的蛋白质无法体验疏水力，因为脂质双层缺乏水。因此出现了基本的难题：伟大的蛋白质表面在伟大的脂质双层中找到其出色的蛋白质伴侣，以忠实地折叠到本地结构？最近，一种正常同型二聚体Cl-/h+的结构稳定且功能性的单体形式通过在二聚体界面引入色氨酸突变来设计抗胞菌CLC-EC1。初步的研究表明，蛋白质可以通过其他突变或某些脂质转移回二聚体状态状况。这些结果将CLC-EC1作为研究可逆二聚化的模型，这简化蛋白质折叠过程，同时仍包含蛋白质的所有热力学特性膜环境中的相互作用。为了进行这些充满活力的测量，一个月/二聚体人口将使用三种公认的方法进行量化：（i）poisson-counting“ vs。脂质体种群中的二聚体，（ii）脂质体中的荧光自我震动，（iii）f rster共振脂质体和支持双层的能量转移（FRET）进行单分子研究。这些测定法在位，将进行实验，以研究两个遍布的替代假设在这个领域的话语。首先，特定的跨膜螺旋相互作用是由van der驱动的 WAALS在高度互补的表面上有力。自由能的变化将在取代时测量对丙氨酸或色氨酸抗界面的耐药性，通过增加侧面 - 链体积调节范德华相互作用。第二个假设是相互作用是在螺旋结合时脂质熵增加的驱动。为了研究这一点，形成脂质的分子溶剂将通过更改化学头组，链长和链顺序来修改溶剂来自古细菌的不饱和或四乙醚脂质。对于所有实验，自由能之间的关系也将是根据温度测量以推断焓和熵的值。这些结果将会提供有关膜蛋白相互作用的驱动力的见解，甚至可以提供基础为了攻击蛋白质折叠的一般问题，即脂质双层。