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

摘要 蛋白质折叠的中心谜题在于自然界的物理力量如何驱动一串简单的氨基 酸变成一种稳定的、构象确定的蛋白质。对于可溶性蛋白质，疏水基团的埋葬 远离水界面是主要的驱动力，但包埋在膜上的蛋白质无法经历 疏水作用力，因为脂质双层缺乏水。这样就产生了一个根本的难题：一个 油腻的蛋白质表面在油腻的脂类双层中找到其油腻的蛋白质伙伴，忠实地折叠成其天然的 结构？最近，一种结构稳定的功能单体形式的正常同二聚氯/H+ 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 研究表明，蛋白质可以通过额外的突变或在某些脂类中转变回二聚体状态。 条件。这些结果表明，ClC-EC1可作为研究可逆二聚反应的模型。 简化了蛋白质折叠过程，同时仍包含蛋白质的所有热力学性质 膜环境中的相互作用。为了进行这些能量测量，单体/二聚体 将使用三种成熟的方法对种群进行量化：(I)单体的泊松计数与 脂质体中的二聚体，(Ii)脂质体中的荧光自猝灭，和(Iii)F？rster共振 用于单分子研究的脂质体和支撑双层中的能量转移(FRET)。通过这些化验 在适当的地方，将进行实验，以调查两个普遍存在的替代假设 这一领域的论述。首先，特定的跨膜螺旋相互作用是由范德焓驱动的。 在高度互补的曲面上的Waals力。自由能的变化将在替代时被测量 丙氨酸或色氨酸的界面残基，通过增加侧基来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 由螺旋缔合时增加的脂类熵驱动。为了研究这一点，形成脂质的分子 通过改变化学头基团、链长和链序对溶剂进行改性 古生菌中的不饱和或四醚类脂类。对于所有实验，自由能关系也将是 相对于温度进行测量，以外推焓和熵的值。这些结果将 洞察膜蛋白相互作用的驱动力，甚至可能提供基础 用来解决蛋白质在奇怪的溶剂中折叠的一般问题，这种奇怪的溶剂就是脂双层。

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.