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Molecular Dynamics And Vibrational Characteristics Of Membrane Assemblies

膜组件的分子动力学和振动特性

基本信息

批准号：
7734016
负责人：
Ira W. Levin
金额：
$ 78.32万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7734016
关键词：
Acoustics Area Behavior Biological Biological Models Calcium ion Cations Cells Cellular Membrane Characteristics Cholesterol Complex DNA Sequence Rearrangement Decompression Sickness Diamond Disease Exhibits Freezing Galactosylceramides Gel Glycosphingolipids Individual Integral Membrane Protein Lateral Lecithin Lipid Bilayers Lipids Liquid substance Magnesium Measurement Measures Melanocytic nevus Membrane Membrane Fusion Membrane Microdomains Modeling Mole the mammal Molecular Monitor Numbers Pattern Phase Phase Transition Phospholipids Property Proteins Raman Spectrum Analysis Research Respiratory Diaphragm Role Route System Techniques Temperature Ultrasonics Velocimetries Vesicle Work carbene cold temperature galactocerebroside infrared spectroscopy insight interest light scattering magnesium ion membrane assembly membrane model molecular dynamics nanoscale nephelometry pressure reconstitution response size sound

项目摘要

Summary of Work: Our research efforts involve the modulatory effects of bilayer lipids on the structural reorganizations of integral membrane proteins. Our interest lay primarily in characterizing the sizes and formation properties of fluctuating lipid microdomains within biomembranes, using vibrational infrared and Raman spectroscopy ultrasonic velocimetry techniques. In particular, the compressibilities of systems composed of various lipid microdomains were correlated with intramolecular protein rearrangements. Various reconstituted multilamellar and single shell vesicle assemblies were generated as model systems to demonstrate the effects arising from the lateral compressibility properties of these quantified lipid microaggregates. To study spectroscopically specific bilayer lipid chain order/disorder properties within the membrane microdomains, appropriate lipid acyl chain deuteration was required to allow the vibrational dynamics of the chain moieties to be monitored. Binary mixtures of saturated chain phosphatidylcholines were specifically examined. Various spectroscopic splitting patterns of the methylene bending modes allowed a determination of lipid microdomain size in terms of the number of acyl chains constituting a given lipid cluster. The compressibilities of the lipid assemblies were determined both isothermally and adiabatically. An infrared diamond anvil cell was used to measure bilayer isothermal compressibility. Pressures were defined by monitoring the spectra of a pressure transducing material, while volume changes were measured directly. Adiabatic compressibilities of the lipid dispersions were determined by ultrasonic velocimetry in which the thermotropic response to the velocity of sound is measured. In examining binary lipid mixtures, microdomain sizes were found to be functions of the lipid mole fractions constituting the system. Specifically, the lateral compressibilities of the binary systems and integral membrane protein reorganizations were governed by the effective domain sizes defining the assembly. A variety of light scattering studies were also performed on single shell vesicle systems in efforts to correlate size with bilayer microdomain properties as a function of temperature. Results were also obtained which demonstrated the use of vibrational infrared spectroscopy applied toward characterizing lipid microdomain sizes derived from a model raft system consisting of non-hydroxy galactocerebroside, cholesterol , and dipalmitoylphosphatidylcholine components. The resulting spectroscopic correlation field components of the lipid acyl chain CH2 methylene deformation modes, observed when lipid multilamellar assemblies are rapidly frozen from the liquid crystalline state to the gel phase, indicated the existence of lipid microdomains at the several nanometer scale. The addition of cholesterol disrupts the glycosphingolipid selectively, in contrast to perturbing the disaturated chain phospholipid matrix. The sizes of the aggregates were determned from the correlation field effects of interacting acyl chains at low temperatures. Complementary acoustic velocimetry measurements indicated that the microdomain formation decreases the total volume adiabatic compressibilities of the multilamellar vesicle assemblies. Addition of cholesterol, however, disrupts the galactocerebroside domains, resulting in a slight increase in the lipid assemblies total adiabatic compressibility. The combination of these two physical approaches offers new insights into microdomain formation and their properties in model bilayer systems. For understanding more completely the steps involved in the transition of two contiguous bilayers as they fuse under the influence of a fusogenic agent, such as the magnesium ion, we emphasize the use of infrared spectroscopic techniques for a detailed characterization of lipid bilayer fusion properties. In particular, we examined the binary DPPS/DPPC bilayer system both to assess lipid microdomain formation and acyl chain rearrangements within the membranes hydrophobic core. In this system, microdomains are distributed throughout the bilayer. In the presence of the magnesium cation , DPPS maintains a gel phase configuration above the phase transition, while DPPC exhibit no response other than to rearrange their acyl chains in a manner consistent with the DPPS matrix. In summary, the lipid reorganizations within predominantly DPPS microdomains represent a critical aspect for disrupting the critical fusion intermediate, the hemifusion diaphragm, in route to complete membrane fusion.

工作概述：我们的研究主要涉及双层脂质对整体膜蛋白结构重组的调节作用。我们的兴趣主要在于表征生物膜内波动脂质微域的大小和形成特性，使用振动红外和拉曼光谱超声测速技术。特别是，由各种脂质微结构域组成的系统的可压缩性与分子内蛋白质重排相关。各种重组的多层和单壳囊泡组装被生成作为模型系统，以证明这些量化的脂质微聚集体的侧向压缩性特性所产生的影响。为了研究膜微域内光谱特异性双层脂质链有序/无序特性，需要适当的脂质酰基链氘化以监测链段的振动动力学。对饱和链磷脂酰胆碱的二元混合物进行了专门的研究。亚甲基弯曲模式的各种光谱分裂模式允许根据构成给定脂质簇的酰基链的数量来确定脂质微结构域的大小。脂质组件的可压缩性采用等温和绝热两种方法测定。采用红外金刚石砧细胞测量双层等温压缩率。通过监测压力传感器材料的光谱来定义压力，而直接测量体积变化。脂质分散体的绝热压缩性是用超声波测速法测定的，其中测量了对声速的热致性响应。在检查二元脂质混合物时，发现微畴大小是构成系统的脂质摩尔分数的函数。具体来说，二元体系和整体膜蛋白重组的横向可压缩性是由定义组装的有效结构域大小决定的。各种光散射研究也在单壳囊泡系统上进行，以努力将尺寸与双层微畴性质作为温度的函数联系起来。研究结果还表明，振动红外光谱可用于表征由非羟基半乳糖脑苷、胆固醇和双棕榈酰磷脂酰胆碱成分组成的模型筏系统的脂质微畴大小。当脂质多层组装体从液晶状态快速冻结到凝胶相时，所得到的脂质酰基链CH2亚甲基变形模式的光谱相关场分量表明，在几个纳米尺度上存在脂质微畴。与干扰不饱和链磷脂基质相反，胆固醇的加入选择性地破坏鞘糖脂。在低温下，通过相互作用的酰基链的相关场效应来确定聚集体的大小。互补声速测量表明，微畴的形成降低了多层囊泡组合的总体绝热压缩率。然而，胆固醇的加入破坏了半乳糖脑苷结构域，导致脂质组合的总绝热压缩性略有增加。这两种物理方法的结合为模型双层系统中的微畴形成及其性质提供了新的见解。为了更全面地理解两个相邻的双分子层在融合剂（如镁离子）的影响下融合时的转变过程，我们强调使用红外光谱技术来详细表征脂质双分子层的融合特性。特别是，我们研究了二元DPPS/DPPC双层体系，以评估膜疏水核心内脂质微结构域的形成和酰基链的重排。在该系统中，微畴分布在整个双层结构中。在镁离子的存在下，DPPS保持了相变以上的凝胶相构型，而DPPC除了以与DPPS基质一致的方式重新排列其酰基链外，没有表现出任何反应。综上所述，DPPS微域内的脂质重组是破坏关键融合中间体（半融合隔膜）以完成膜融合的关键方面。