Molecular Dynamics And Vibrational Characteristics Of Membrane Assemblies

工作总结：我们的研究工作涉及双层脂质对膜蛋白结构重组的调节作用。 我们的兴趣在于主要是在生物膜内的波动脂质微区的大小和形成特性的特征，使用振动红外和拉曼光谱超声测速技术。特别地，由各种脂质微区组成的系统的压缩与分子内蛋白质重排相关。 各种重建的多层和单壳囊泡组件生成的模型系统，以证明这些量化的脂质微聚集体的横向压缩性能所产生的影响。 为了研究光谱特定的双层脂链有序/无序特性的膜微域内，需要适当的脂酰基链氘化，以允许振动动力学的链部分进行监测。具体检查了饱和链磷脂酰胆碱的二元混合物。各种光谱分裂图案的亚甲基弯曲模式允许的脂质微区的大小的确定方面的酰基链构成一个给定的脂质簇的数量。在等温和等温两种条件下测定了脂质组装体的压缩率。 用红外金刚石压砧装置测量了双层膜的等温压缩系数。 通过监测压力传感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率通过超声测速法测定，其中测量对声速的热致响应。在检查二元脂质混合物，微区的大小被发现构成系统的脂质摩尔分数的函数。具体而言，横向压缩的二元系统和完整的膜蛋白重组的有效域的大小定义的组装。各种光散射的研究也进行了单壳囊泡系统的努力，以关联大小与双层微区的性能作为温度的函数。还获得了结果，证明了使用振动红外光谱法应用于表征来自模型筏系统的非羟基半乳糖苷，胆固醇，和二棕榈酰磷脂酰胆碱组分的脂质微区的大小。 脂质酰基链CH 2亚甲基变形模式的光谱相关场分量，观察到脂质多层组装时，迅速冻结从液晶状态的凝胶相，表明在几个纳米尺度的脂质微区的存在。 胆固醇的加入选择性地破坏鞘糖脂，而不是扰动二饱和链磷脂基质。聚集体的尺寸由低温下相互作用的酰基链的相关场效应确定。互补的声速测量表明，微区的形成减少了多层囊泡组件的总体积绝热压缩。 然而，胆固醇的加入破坏了半乳糖苷的结构域，导致脂质组装体的总绝热压缩性略有增加。 这两种物理方法的结合为微区的形成及其在模型双层系统中的性质提供了新的见解。 为了更全面地了解两个相邻的双层的过渡所涉及的步骤，因为它们融合的影响下的融合剂，如镁离子，我们强调使用红外光谱技术的详细表征的脂质双层融合特性。 特别是，我们研究了二元DPPS/DPPC双层系统，以评估膜疏水核心内的脂质微区形成和酰基链重排。 在该系统中，微区分布在整个双层中。 在镁阳离子的存在下，DPPS在相变之上保持凝胶相构型，而DPPC除了以与DPPS基质一致的方式重排其酰基链之外没有表现出响应。 总之，主要DPPS微域内的脂质重组代表了在完成膜融合的途径中破坏关键融合中间体半融合隔膜的关键方面。

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.