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

描述(申请人提供)：蛋白质折叠的中心谜团在于自然的物理力量如何将一串简单的氨基酸转化为稳定的、构象定义的蛋白质。对于可溶性蛋白质来说，疏水基团被埋入远离水界面是主要的驱动力，但包埋在膜上的蛋白质不能经历疏水性，因为脂质双层缺乏水。一个根本的难题由此产生：一个油腻的蛋白质表面如何在油腻的脂类双层中找到它的油腻蛋白质伙伴，以忠实地折叠到它的自然结构中？最近，一种结构稳定、功能齐全的单体形式 在正常的同源二聚体中，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质条件下恢复到二聚体状态。这些结果表明，CLC-EC1是一个研究可逆二聚的模型，它简化了蛋白质折叠过程，同时仍然包含了膜环境中蛋白质相互作用的所有热力学性质。为了进行这些能量测量，将使用三种成熟的方法对单体/二聚体群体进行量化：(I)脂质体群体中单体和二聚体的泊松计数；(Ii)脂质体中的荧光自猝灭；以及(Iii)单分子研究中脂质体和支撑双层中的Forster共振能量转移(FRET)。有了这些分析，实验将被用来研究这一领域中普遍存在的两种可供选择的假设。首先，这种特定的跨膜螺旋相互作用是由高度互补表面的范德华力所驱动的。自由能的变化将在界面残基被取代为丙氨酸或色氨酸时被测量，重要的位置将通过增加侧链体积来调节范德华相互作用来进一步研究。第二个假设是，相互作用是由螺旋结合时增加的脂类熵驱动的。为了研究这一点，形成脂类溶剂的分子将通过使用古生菌中的不饱和或四醚类脂改变化学头基、链长和链顺序来进行修饰。对于所有的实验，还将测量自由能与温度的关系，以外推热和熵的值。这些结果将提供对驾驶的洞察 对于膜蛋白相互作用的作用力，甚至可能为解决蛋白质在奇怪的溶剂中折叠的一般问题提供基础，这种奇怪的溶剂就是脂双层。 与公共健康相关：膜蛋白是调节生物材料通过脂质双层的分子“守门人”。因此，他们关键地参与了 在生理过程中，并可能是关键的治疗靶点。通过了解控制这些蛋白质如何在脂质环境中相互作用和组装的能量因素，我们将深入了解调控膜蛋白功能和细胞生理的方法。

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. PUBLIC HEALTH RELEVANCE: Membrane proteins are molecular "gate-keepers" regulating the passage of biological materials across the lipid bilayer. As such, they are critically involved in physiological processes and may be key therapeutic targets. By understanding the energetic factors governing how these proteins interact and assemble in the lipid environment, we will gain insight into methods of modulating membrane protein function and cell physiology.