Driving forces of membrane protein assembly in membranes

摘要 影响膜蛋白折叠自由能的热力学驱动力是什么？ 脂类双层中的组装？对于可溶性蛋白质，疏水基团的掩埋远离水 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力量， 因为脂质双层缺乏水分。一个根本的难题由此产生：油腻的蛋白质表面是如何找到的 它的油腻的蛋白质在油腻的脂类双层中忠实地折叠成它的天然结构？最近，一位 结构稳定且具有功能的单体形式的正常均二聚体Cl-/H+逆向转运蛋白ClC-EC1是 通过在二聚体界面引入色氨酸突变来设计。我们已经利用这一点开发了一种新的 用于自由能测量的膜中可逆二聚反应研究的模型系统 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。要在广泛的范围内量化单体和二聚体的数量 根据蛋白质/脂的摩尔比，我们发展了(I)Förster共振能量转移(FRET)和(Ii)单 脂质体中分子光漂白的全内反射显微镜方法。 单分子显微镜的灵敏度使我们可以在极稀薄的条件下观察。 ClC-EC1在膜中的解离。随着能量学的测量已经到位，我们将 调查这一领域中普遍存在的两种可供选择的假设。首先，蛋白质之间的联系 是由高度互补表面上的范德华力驱动的。自由能的变化将是 根据界面残基取代丙氨酸或色氨酸进行测量，并努力确定VDW是否 主题可以被赋予已经不稳定的结构。第二个假设是，相互作用是 由亚基结合时增加的脂类熵驱动。为了研究这一点，形成脂质的分子 将通过测试疏水不匹配作为酰基链长的函数来修饰溶剂，并且 通过改变脂类的回转半径来耗尽吸力，例如，更大的不饱和和四醚类脂类。 较小的非极性全身麻醉药。对于所有实验，自由能关系将以 温度对热变和熵变的外推作用。这项研究将由一名 罗伯逊实验室的跨学科科学家团队，受过研究生的培训， 博士后，研究科学家和首席研究员，结合膜蛋白生物化学的专业知识， 单分子显微镜和计算模型提供了对此的无限研究 重要的生物物理问题。这项研究的结果将为 旨在纠正蛋白质错误折叠或调节蛋白质相互作用的知情策略的发展 在生理和病理情况下的膜。

ABSTRACT What are the thermodynamic driving forces that influence the free energy of membrane protein folding and assembly in lipid bilayers? 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. We have used this to develop a new model system for studying reversible dimerization in membranes for free energy measurements, which simplifies the protein folding process while still encompassing all of the thermodynamic properties of protein interactions in the membrane environment. To quantify monomer vs. dimer populations across a wide range of protein per lipid mole ratios, we developed (i) Förster resonance energy transfer (FRET) and (ii) single- molecule photo bleaching by total internal reflection microscopy in liposomes methods for the CLC-ec1 system. The sensitivity of single-molecule microscopy allows us to go to extremely dilute conditions where we observe dissociation of CLC-ec1 in membranes. With measurements of the energetics already in place, we will investigate two alternative hypotheses that have pervaded discourse in this field. First, that protein association is 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, and efforts made to identify if VDW motifs can be conferred to already destabilized constructs. The second hypothesis is that interactions are driven by increased entropy of lipids upon subunit association. To study this, the molecules forming the lipid solvent will be modified by testing hydrophobic mismatch as a function of acyl chain length, and also the depletion-attraction force by changing lipid radius of gyration, e.g. larger unsaturated and tetraether lipids vs. smaller non-polar general anesthetics. For all experiments, free energy relationships will be measured as a function of temperature to extrapolate enthalpy and entropy changes. This research will be carried out by a team of interdisciplinary scientists in the Robertson laboratory, with levels of training from graduate student, postdoc, research scientist and principal investigator, combining expertise of membrane protein biochemistry, single-molecule microscopy and computational modeling to provide an unlimited investigation into this important biophysical question. The results from this study will provide a physical foundation for the development of informed strategies aimed at correcting protein mis-folding or regulating protein interactions in membranes in physiologically and pathological situations.