Molecular Dynamics And Vibrational Characteristics Of Membrane Assemblies

工作总结：我们的研究工作涉及双层脂类对完整膜蛋白结构重组的调节作用。我们的兴趣主要集中在利用振动红外和拉曼光谱超声测速技术来表征生物膜内波动的脂质微域的大小和形成特性。特别是，由不同脂质微区组成的体系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件被生成作为模型系统，以演示这些量化的脂质微团聚体的横向可压缩性所产生的影响。为了研究膜微区内特定的双层脂链的有序/无序性质，需要适当的脂酰链氢化，以便能够监测链部分的振动动力学。特别研究了饱和链磷脂酰胆碱的二元混合物。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簇的酰基链的数量来确定脂微结构域大小。用等温法和绝热法测定了脂类集合体的压缩系数。用红外金刚石压腔测量了双分子层的等温压缩系数。压力是通过监测压力传感材料的光谱来定义的，而体积变化是直接测量的。用超声波测速仪测定了脂类分散体的绝热压缩系数，测量了其对声速的热致响应。在检查二元脂类混合物时，发现微区大小是构成体系的脂摩尔分数的函数。具体地说，二元体系和完整膜蛋白重组的横向可压缩性由定义组装的有效结构域大小控制。在单层囊泡系统上也进行了各种光散射研究，以努力将大小与双层微域性质作为温度的函数相关联。研究结果还表明，振动红外光谱技术可用于表征由非羟基半乳脑苷脂、胆固醇和二棕榈酰磷脂酰胆碱组成的模型RAFT体系中的脂质微区大小。由此得到的脂酰链CH2亚甲基变形模式的光谱关联场分量，当脂类多层组装从液晶态快速冻结到凝胶相时观察到，表明在几个纳米尺度上存在脂类微区。与扰乱饱和的链状磷脂基质相反，胆固醇的加入选择性地破坏了神经鞘糖脂。聚集体的大小由低温下相互作用的酰基链的关联场效应确定。互补声速测量表明，微区的形成降低了多层囊泡组件的总体积绝热压缩系数。然而，胆固醇的添加扰乱了半乳脑苷域，导致脂类总绝热可压缩性略有增加。这两种物理方法的结合为模型双层体系中微区的形成及其性质提供了新的见解。为了更全面地了解两个连续的双层在融合剂(如镁离子)的影响下融合时所涉及的步骤，我们强调使用红外光谱技术来详细表征脂质双层的融合性质。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域的形成和酰链重排。在该系统中，微区分布在整个双层中。在镁离子存在的情况下，DPPS在相变上方保持凝胶相构型，而DPPC除了以与DPPS基质一致的方式重排它们的酰基链外，没有表现出任何反应。综上所述，以DPPS为主的微域内的脂质重组是破坏关键的融合中间体--半灌注膜，从而完成膜融合的关键方面。

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.