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和超声测速技术。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。用超声波测速法测定了脂质分散体的绝热压缩率，其中测量了对声速的热致响应。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。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Mg（2+）的存在下，DPPS通过相变温度保持有序的正交晶系亚胞凝胶相，而DPPC组分相对于凝胶到液晶相变仅受到最小的扰动。DPPC矩阵没有表现出响应以外，以符合DPPS矩阵的方式重新排列其酰基链。 Mg（2+）的加入诱导了脂质结构域的重组，其中凝胶相acy链平面从六方构型向三斜平行链亚胞重排。 在低温下的酰基链亚甲基变形模式的检查允许DPPS微区尺寸的测定，其在不存在镁离子的情况下在添加DPPC时减少。 加入Mg（2+）后，在二元混合物中观察到了均匀的DPPS尺寸。在该系统中，微区分布在整个双层中。光散射和荧光测量结果表明，镁诱导的聚集和融合的脂质组件作为一个功能的DPPS的DPPC的比例。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重要机制方面。

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 spectroscopies and 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. Adiabatic compressibilities of 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. 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 a membranes hydrophobic core. In the presence of Mg(2+) DPPS maintains an ordered orthorhombic subcell gel phase though the phase transition temperature, while the DPPC component is only minimally perturbed with respect to the gel to liquid crystalline phase change. The DPPC matrix exhibits no response other than to rearrange its acyl chains in a manner consistent with the DPPS matrix. The addition of Mg(2+) induces a reorganization of the lipid domains in which the gel phase acy chain planes rearrange from an hexagonal configuration toward a triclinic, parallel chain subcell. Examination of the acyl chain methylene deformation modes at low temperatures allows a determination of DPPS microdomain sizes, which decrease upon the addition of DPPC in the absence of the magnesium ion. On adding Mg(2+), a uniform DPPS size is observed in the binary mixtures. In this system, microdomains are distributed throughout the bilayer. Light scattering and fluorescence measurements indicated that magnesium induces both the aggregation and the fusion of the lipid assemblies as a function of the ratio of DPPS to DPPC. In summary, the lipid reorganizations within predominantly DPPS microdomains represent a mechanistically important aspect for disrupting the critical fusion intermediate, the hemifusion diaphragm, in route to complete membrane fusion.