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

描述（由申请人提供）：蛋白质折叠的核心谜在于自然界的物理力量如何将一串简单的氨基酸驱动成稳定的、构象确定的蛋白质。对于可溶性蛋白来说，疏水性基团远离水界面的埋藏是一个主要的驱动力，但由于脂质双分子层缺乏水，膜嵌入的蛋白质不能经历疏水力。因此，一个基本的难题出现了：一个油腻的蛋白质表面如何在油腻的脂质双分子层中找到它的油腻蛋白质伙伴，并忠实地折叠成它的天然结构？最近，一种结构稳定且具有功能的单体形式出现

DESCRIPTION (provided by applicant): 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) Forster 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.