Microstructural Heterogeneity in Membranes

膜的微观结构异质性

• 批准号: 7869486
• 负责人: Barry R Lentz
• 金额: $ 9.88万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-10 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7869486
• 关键词: Address; Behavior; Calcium-Binding Proteins; Canis familiaris; Cell membrane; Cell physiology; Cells; Chimeric Proteins; Cholesterol; Cholesterol Esters; Complex; Cytoplasmic Tail; DNA Sequence Rearrangement; Defect; Electron Microscopy; Electrons; Ethylene Glycols; Extravasation; Fluorescence; Free Energy; Freeze Fracturing; Funding; HIV; HIV Envelope Protein gp41; Heterogeneity; Infection; Influenza; Influenza Hemagglutinin; Kinetics; Lead; Life Cycle Stages; Lipid Bilayers; Lipids; Maleimides; Measures; Mechanics; Mediating; Membrane; Membrane Fusion; Modeling; Monitor; Mutation; NMR Spectroscopy; Neurons; Peptides; Phase; Phosphatidylinositol 4,5-Diphosphate; Polymers; Pore Proteins; Process; Property; Proteins; Pyrenes; Reaction; Recombinants; Reporting; Research Personnel; Respiratory Diaphragm; Spectroscopy, Fourier Transform Infrared; Stabilizing Agents; Stress; Structure; Surface; Synaptic Vesicles; System; Tertiary Protein Structure; Testing; Vesicle; Vesicular stomatitis Indiana virus; Viral; Viral Fusion Proteins; Viral Proteins; Virus; Virus Diseases; Water; Work; X ray diffraction analysis; X-Ray Diffraction; aqueous; base; effusion; ethylene glycol; hexadecane; in vivo; influenzavirus; insight; lipid I; membrane model; monolayer; mutant; neurotransmitter release; peptide structure; promoter; protein function; receptor; reconstitution; research study; solid state nuclear magnetic resonance; synaptotagmin; syntaxin; syntaxin 1; vesicle-associated membrane protein

Regulated membrane fusion is essential to many cell processes and to the life cycle of lipid-sheathed viruses such as HIV, Influenza, and Ebola. Several proteins ("fusion machines") involved in neurotransmitter release and enveloped viral infection have been identified and characterized structurally. Still, how these proteins catalyze fusion is not fully understood. The Lentz lab studies lipid rearrangements associated with fusion between synthetic membranes. The approach is first to define these rearrangements in pure lipid systems and then to ask how fusion proteins might promote them. Fusion requires close contact between membranes, which is induced using the inert polymer poly(ethylene glycol) (PEG). Fusion between lipid vesicles aggregated by PEG is shown to be minimally a three-step process (contacted bilayers D "stalk" intermediate D "diaphragm" intermediate D pore) that mimics biomembrane fusion. The Lentz group calculated, using the mechanical properties of lamellar lipid phases, the free energy reaction profile of this "stalk" fusion mechanism and showed it to be consistent with their unique studies effusion kinetics. Experiments and calculations show that the free energies of bent lipid monolayers and of defects between non-lamellar and lamellar structures (hydrophobic interstices) dominate the fusion process. Most researchers have focused on the bending energy to explain fusion. The Central Hypothesis is that fusion proteins catalyze fuSion not just by altering bending energy but in good measure by stabilizing hydrophobic interstices. To test this hypothesis, the project will address seven Specific Aims: 1] Compare the abilities of different bend-inducing lipids to partition into or promote bent lipid structures, and 2] alter the fusion reaction mechanism; 3] Determine whether an infection-blocking mutation in the membrane spanning domain of HIV alters the structure of a synthetic membrane spanning domain peptide, alters membrane structure, and alters the effect of this peptide on fusion; 4] Determine whether mutations in a key region of Influenza virus (the "fusion" peptide) alter a} the structure of a synthetic fusion peptide, b} membrane structure, and c} the effect of this peptide on fusion; 5] Determine whether neurotransmitter- release-blocking mutations in the membrane-spanning region of a neuronal fusion protein (syntaxin) alter a} the structure of a synthetic membrane spanning domain peptide, b} membrane structure, and c} the effect of this peptide on fusion; 6] Determine by electron microscopy whether fusion pores form at the point of contact between membranes held in contact by neuronal fusion proteins; and 7] Determine the ability of a neuronal calcium-binding protein (synaptotagmin) to perturb, and trigger fusion between, model membranes brought

受调控的膜融合对许多细胞过程和脂质鞘病毒的生命周期至关重要 如艾滋病、流感和埃博拉病毒。参与神经递质释放的几种蛋白质（“融合机器”） 和包膜病毒感染已经被鉴定并在结构上表征。尽管如此，这些蛋白质 催化聚变尚未完全理解。Lentz实验室研究与融合相关的脂质重排 在合成膜之间。该方法首先是在纯脂质体系中定义这些重排 然后研究融合蛋白是如何促进它们的。融合需要密切联系， 膜，这是诱导使用惰性聚合物聚（乙二醇）（PEG）。脂质间融合 聚乙二醇聚集的囊泡被证明是一个最低限度的三步过程（接触双层D“茎” 中间D“隔膜”中间D孔）。Lentz集团 计算，使用层状脂质相的机械性质，该脂质相的自由能反应曲线。 “茎”融合机制，并表明这是符合他们独特的研究渗出动力学。 实验和计算表明，弯曲脂质单分子膜的自由能和 非层状和层状结构（疏水间隙）主导熔融过程。最 研究者们把注意力集中在弯曲能量上来解释核聚变。中心假设是核聚变 蛋白质催化聚变不仅通过改变弯曲能，而且在很大程度上通过稳定疏水性 空隙为了验证这一假设，该项目将涉及七个具体目标： 1]比较不同弯曲诱导脂质分配到或促进弯曲脂质结构的能力，以及 2]改变融合反应机制; 3]确定是否存在感染阻断突变， HIV的跨膜结构域改变了合成的跨膜结构域肽的结构， 改变膜结构，并改变这种肽对融合的影响; 4]确定是否突变， 流感病毒的关键区域（“融合”肽）改变a}合成融合肽的结构，B} 膜结构，和c}该肽对融合的影响; 5]确定神经递质- 神经元融合蛋白（突触融合蛋白）跨膜区的释放阻断突变改变了 合成的跨膜结构域肽的结构，B}膜结构，和c} 通过电子显微镜确定在接触点是否形成融合孔 在通过神经元融合蛋白保持接触的膜之间;和7]确定神经元融合蛋白的能力。 钙结合蛋白（synaptotagmin）干扰，并触发融合之间，模型膜带来