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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.

摘要