Driving forces of membrane protein assembly in membranes

膜蛋白在膜中组装的驱动力

基本信息

批准号：
9324291
负责人：
Janice L Robertson
金额：
$ 33.16万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-01 至 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9324291
关键词：
Affect Alanine Amino Acid Substitution Arachidonic Acids Biological Models Biophysics Burial CLC Gene Cell membrane Cellular biology Chloroform Complex Computer Simulation Crystallization Development Dimerization Disease Dissociation Educational Status Energy Transfer Engineering Entropy Environment Equilibrium Escherichia coli Fluorescence Microscopy Foundations Free Energy General anesthetic drugs Goals Gray unit of radiation dose Hydrophobicity Investigation Laboratories Length Lipid Bilayers Lipids Liposomes Maps Measurement Measures Membrane Membrane Lipids Membrane Proteins Membrane Transport Proteins Methods Microscopy Modeling Mole the mammal Mutation Outcomes Research Pathologic Photobleaching Physiological Physiology Play Population Postdoctoral Fellow Principal Investigator Process Property Protein Biochemistry Protein Subunits Proteins Quality Control Radial Research Role Scientist Side Solvents Structure Study models Surface System Temperature Testing Thermodynamics Tryptophan Water Work Xenon antiporter aqueous crosslink density design dimer driving force enthalpy experience experimental study graduate student monomer protein folding protein misfolding reconstitution single molecule van der Waals force

Affect Alanine Amino Acid Substitution Arachidonic Acids Biological Models Biophysics Burial CLC Gene Cell membrane Cellular biology Chloroform Complex Computer Simulation Crystallization Development Dimerization Disease Dissociation Educational Status Energy Transfer Engineering Entropy Environment Equilibrium Escherichia coli Fluorescence Microscopy Foundations Free Energy General anesthetic drugs Goals Gray unit of radiation dose Hydrophobicity Investigation Laboratories Length Lipid Bilayers Lipids Liposomes Maps Measurement Measures Membrane Membrane Lipids Membrane Proteins Membrane Transport Proteins Methods Microscopy Modeling Mole the mammal Mutation Outcomes Research Pathologic Photobleaching Physiological Physiology Play Population Postdoctoral Fellow Principal Investigator Process Property Protein Biochemistry Protein Subunits Proteins Quality Control Radial Research Role Scientist Side Solvents Structure Study models Surface System Temperature Testing Thermodynamics Tryptophan Water Work Xenon antiporter aqueous crosslink density design dimer driving force enthalpy experience experimental study graduate student monomer protein folding protein misfolding reconstitution single molecule van der Waals force

展开

项目摘要

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.

抽象的什么是影响膜蛋白折叠和的自由能的热力学驱动力和脂质双层组装？对于固体蛋白，疏水基团的埋葬接口是主要的驱动力，但是膜包裹的蛋白质无法体验疏水力，由于脂质双层缺乏水。因此出现了基本的难题：伟大的蛋白质表面如何找到它在伟大的脂质双层中的伟大蛋白质伴侣忠实地折叠成本地结构？最近，正常同型二聚体Cl-/h+抗胞菌CLC-EC1的结构稳定和功能性单体形式通过在二聚体界面引入色氨酸突变来设计。我们已经用它来开发一个新的用于研究自由能测量机制可逆二聚化的模型系统，这些系统简化蛋白质折叠过程，同时仍包含蛋白质的所有热力学特性膜环境中的相互作用。量化单体与二聚体种群蛋白质每个脂质分子比，我们开发了（i）förster共振能量转移（FRET）和（ii） CLC-EC1系统的脂质体方法中的总内反射显微镜通过总内反射显微镜进行了光漂白。单分子显微镜的敏感性使我们能够进入极稀释的条件，我们观察到 Clc-ec1在膜中的解离。随着已经到位的能量学的测量，我们将研究两个在该领域中遍布话语的替代假设。首先，蛋白质关联是由高度互补表面上的范德华力驱动的。自由能的变化将是用居住在丙氨酸或色氨酸的界面取代的情况下测量，并努力确定是否VDW 图案可以赋予已经不稳定的结构。第二个假设是相互作用是在亚基关联时脂质的熵增加而驱动。为了研究这一点，形成脂质的分子溶剂将通过测试疏水不匹配作为酰基链长度的函数来改变溶剂通过改变回旋的脂质半径，例如较大的不饱和和四脂脂质与。较小的非极性通用麻醉药。对于所有实验，自由能关系将被衡量温度的功能以推断焓和熵变化。这项研究将由罗伯逊实验室的跨学科科学家团队，研究生的培训水平博士后，研究科学家和主要研究者，结合了膜蛋白生物化学专业知识，单分子显微镜和计算建模，以对此进行无限研究重要的生物物理问题。这项研究的结果将为开发旨在纠正蛋白质折叠或调节性蛋白质相互作用的知情策略身体和病理情况下的膜。