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Microstructural Heterogeneity in Membranes

膜的微观结构异质性

基本信息

批准号：
7893100
负责人：
Barry R Lentz
金额：
$ 32.23万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-12-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7893100
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

项目摘要

DESCRIPTION (provided by applicant): 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 also 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

描述（由申请人提供）：受调节的膜融合对许多细胞过程和脂质鞘病毒（如HIV、流感和埃博拉病毒）的生命周期至关重要。一些参与神经递质释放和包膜病毒感染的蛋白质（“融合机器”）已经被确定并在结构上进行了表征。然而，这些蛋白质是如何催化融合的还没有被完全理解。Lentz实验室研究与合成膜融合相关的脂质重排。方法是首先在纯脂质系统中定义这些重排，然后询问融合蛋白如何促进它们。融合需要膜之间的紧密接触，这是用惰性聚合物聚乙二醇（PEG）诱导的。聚乙二醇聚集的脂质囊泡之间的融合被证明是一个最小的三步过程（接触双层D“茎”中间D“隔膜”中间D孔），模拟生物膜融合。Lentz小组利用层状脂相的力学性质计算了这种“茎”融合机制的自由能反应谱，并表明其与他们独特的研究相一致。实验和计算表明，弯曲的脂质单层和非层状和层状结构之间的缺陷（疏水间隙）的自由能在融合过程中起主导作用。大多数研究人员都集中在弯曲能上来解释核聚变。核心假设是融合蛋白催化融合不仅通过改变弯曲能量，而且在很大程度上通过稳定疏水间隙。为了验证这一假设，该项目将解决七个具体目标：1]比较不同的弯曲诱导脂质分裂成或促进弯曲脂质结构的能力；2]改变融合反应机制；3]确定HIV跨膜结构域的感染阻断突变是否改变了合成的跨膜结构域肽的结构，改变了膜结构，并改变了该肽对融合的影响；4]确定流感病毒关键区域（“融合”肽）的突变是否改变a}合成融合肽的结构，b}膜结构，以及c}该肽对融合的影响；5]确定神经融合蛋白（syntaxin）跨膜区域的神经递质释放阻断突变是否会改变a}合成的跨膜结构域肽的结构，b}膜结构，c}该肽对融合的影响；6]通过电子显微镜确定融合孔是否在神经元融合蛋白接触的膜之间的接触点形成；[7]确定神经元钙结合蛋白（synaptotagmin）干扰和触发模型膜融合的能力