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， 组装在脂质双层中？对于可溶性蛋白质，疏水基团远离水溶液的掩埋 界面是一个主要的驱动力，但膜包埋蛋白质不能经历疏水力， 因为脂质双层缺乏水。一个基本的难题由此产生：一个油腻的蛋白质表面是如何找到 它的油脂蛋白质伴侣在油脂脂质双层折叠忠实地进入其天然结构？近日一 结构稳定和功能单体形式的正常同型二聚体Cl-/H+反向转运蛋白CLC-ec 1是 通过在二聚体界面引入色氨酸突变而设计。我们用它来开发一种新的 用于自由能测量的研究膜中可逆二聚作用的模型系统， 简化了蛋白质折叠过程，同时仍然包含蛋白质的所有热力学性质 膜环境中的相互作用。为了在广泛的范围内定量单体与二聚体群体， 蛋白质/脂质摩尔比，我们开发了（i）福斯特共振能量转移（FRET）和（ii）单- 分子光漂白的全内反射显微镜在脂质体方法的CLC-ec 1系统。 单分子显微镜的灵敏度使我们能够在极稀的条件下观察 CLC-ec 1在膜中的解离。随着能量学的测量已经到位，我们将 调查在这个领域中已经普遍存在的两个可供选择的假设。首先，蛋白质的结合 在高度互补的表面上由货车范德华力驱动。自由能的变化 在界面残基被丙氨酸或色氨酸取代后测量，并努力鉴定VDW是否 基序可以被赋予已经不稳定的构建体。第二个假设是， 由亚基缔合后脂质的熵增加驱动。为了研究这个，形成脂质的分子 溶剂将通过测试作为酰基链长度的函数的疏水性错配来进行修改，并且还将通过测试作为酰基链长度的函数的疏水性错配来修改溶剂。 通过改变脂质的回转半径，例如较大的不饱和和四醚脂质与 较小的非极性全身麻醉药。对于所有实验，自由能关系将被测量为 温度外推焓变和熵变的函数。这项研究将由一个 罗伯逊实验室的跨学科科学家团队，从研究生， 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建模提供了一个无限的调查 重要的生物物理问题。本研究的结果将为今后的研究提供物理基础。 制定明智的策略，旨在纠正蛋白质错误折叠或调节蛋白质相互作用， 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.