Microstructural Heterogeneity in Membranes

膜的微观结构异质性

基本信息

批准号：
7448556
负责人：
Barry R Lentz
金额：
$ 32.56万
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 2011-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7448556
关键词：
Address Behavior Calcium-Binding Proteins Canis familiaris Cell membrane Cell physiology Cells Chimeric Proteins Cholesterol Cholesterol Esters Class Complex Cytoplasmic Tail DNA Sequence Rearrangement Decompression Sickness Defect Electron Microscopy Electrons Ethylene Glycols Extravasation Fluorescence Free Energy Freeze Fracturing Funding HIV HIV Envelope Protein gp41 Helix (Snails)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 Rate Reaction Recombinants Reporting Research Personnel Respiratory Diaphragm Spectroscopy, Fourier Transform Infrared Stabilizing Agents Stress Structure Surface Synaptic Vesicles System Tertiary Protein Structure Testing Thinking 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 ear helix effusion ethylene glycol hexadecane in vivo influenzavirus insight lipid I membrane model monolayer mutant neurotransmitter release peptide structure promoter protein function pyrene receptor reconstitution research study solid state 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）：调节膜融合对于许多细胞过程以及脂质鞘病毒（例如 HIV、流感和埃博拉）的生命周期至关重要。几种参与神经递质释放和包膜病毒感染的蛋白质（“融合机”）已被鉴定和结构表征。尽管如此，这些蛋白质如何催化融合尚不完全清楚。 Lentz 实验室研究与合成膜之间融合相关的脂质重排。该方法首先定义纯脂质系统中的这些重排，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，这是使用惰性聚合物聚乙二醇（PEG）诱导的。由 PEG 聚集的脂质囊泡之间的融合至少是一个模仿生物膜融合的三步过程（接触双层 D“茎”中间 D“隔膜”中间 D 孔）。伦茨小组利用层状脂质相的机械特性计算了这种“茎”聚变机制的自由能反应曲线，并表明它与他们独特的渗出动力学研究一致。实验和计算表明，弯曲脂质单层以及非层状和层状结构之间的缺陷（疏水间隙）的自由能主导着融合过程。大多数研究人员都专注于弯曲能来解释聚变。中心假设是融合蛋白不仅通过改变弯曲能来催化融合，而且在很大程度上还通过稳定疏水间隙来催化融合。为了检验这一假设，该项目将解决七个具体目标：1] 比较不同弯曲诱导脂质划分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跨膜结构域中的感染阻断突变是否会改变合成跨膜结构域肽的结构，改变膜结构，并改变该肽对融合的影响； 4] 确定流感病毒关键区域（“融合”肽）的突变是否会改变 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] 确定神经元融合蛋白（突触蛋白）跨膜区域的神经递质释放阻断突变是否会改变 a} 合成跨膜结构域肽的结构、b} 膜结构和 c} 该肽的作用关于融合； 6]通过电子显微镜确定在神经元融合蛋白保持接触的膜之间的接触点处是否形成融合孔； 7]确定神经元钙结合蛋白（突触结合蛋白）扰乱并触发模型膜之间融合的能力