MOLECULAR DYNAMICS AND VIBRATIONAL CHARACTERISTICS OF MEMBRANE ASSEMBLIES

工作总结：我们的研究工作主要包括两个方面：(1)双层脂类对完整膜蛋白结构重组的调制作用；(2)振动拉曼光谱和红外光谱成像技术的仪器开发和应用。(1A)我们对生物膜内波动的脂类微区的影响的研究最近集中在由脂类单链或多不饱和sn-2链和饱和sn-1链组装组成的双层基质中的团簇形成。这些脂类微团聚体的侧向可压缩性有效地对发生在完整膜蛋白内的诱导构象变化施加了调制影响。在研究光谱特定的脂类双层时，适当的酰基氢化使每个链部分的振动动力学能够被单独监测。我们继续使用拉曼光谱和红外光谱技术，结合冷冻猝灭方法，研究由1-eicosanoyl(d39)-2-eicosenoyl-sn-glycero-3-phosphocholine[C(20-d39)：C(20：1 deltaj)PC，其中j=5，8，11和13]组成的双层系列。研究了1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine(POPC-D31)双层膜的酰基链堆积特性和微区形成，确定了与每一类脂类和每一链体系相关的有序/无序参数。用拉曼光谱技术对多晶样品进行了分析，而红外光谱方法则应用于双分子层水分散体。计算技术提供了链簇大小的估计，对于这些不同的磷脂系列，链簇大小在3-19个饱和酰链之间变化。例如，最大的sn-1脂链域，具体起源于相邻分子链之间的van der Waals相互作用，发生在C(20-d39)：C(20：1增量5)PC物种中。使用这些光谱方法，我们还详细地检查了由高度不饱和和完全饱和的脂类组件组成的各种双层组件的微观异质性。(1B)鞘磷脂和胆固醇的膜复合体，或称筏子，最近被认为具有涉及细胞运输、信号转导和各种与膜相关的活动的作用。利用红外光谱技术研究了二棕榈酰磷脂酰胆碱膜双层基质存在和不存在的情况下，非羟基脂肪酸半乳脑苷脂(GalCer)与胆固醇组成的二元和三元混合物的相互作用。在脂凝胶阶段，胆固醇优先作用于GalCer的界面区。此外，胆固醇诱导较长的GalCer链堆积成正交亚细胞簇，支持神经鞘脂膜和质膜中高胆固醇有序相中存在结构域。(2)重点加强了我们的中红外光谱化学成像显微技术，将步进扫描干涉测量与最先进的红外灵敏二维焦平面阵列探测器相结合。将高性能数字成像与非侵入性、高分辨率光学光谱学相结合，可以可视化各种宿主环境中不同化学物种的空间分布。这项技术的力量还体现在同时获取每个空间位置的红外光谱上。这种方法也已扩展到可见光光谱区域，在该区域使用CCD探测器和适当的液晶可调谐滤光片来识别波长，从而获得反射光谱。这种特殊的反射装置已在临床上用于评估烧伤患者皮肤的血液流动。作为该技术在诊断病理学中的应用的例子，从前列腺组织切片和特定皮肤病变(Birt-Hogg-Dube综合征)的切片中获得了有希望的结果。红外吸收和拉曼发射图像，以及它们相关的振动光谱，提供了疾病动物和对照动物未染色组织之间的生化差异的定性描述。对反映组成组织的不同细胞类型的个体光谱的统计分析提供了对观察到的生化变化的简明定量描述。

Summary of Work: Our research efforts encompassed two general areas: (1) The modulatory effects of bilayer lipids on the structural reorganizations of integral membrane proteins, and (2) the instrumental development and applications of vibrational Raman and infrared spectroscopic imaging techniques. (1A) Our interest in characterizing the effects of fluctuating lipid microdomains within biomembranes has recently focused on cluster formation within bilayer matrices comprised of lipid mono- or polyunsaturated sn-2 chain and saturated sn-1 chain assemblies. The lateral compressibility properties of these lipid microaggregates are effective in exerting a modulatory influence on induced conformational changes occurring within integral membrane proteins. In studying spectroscopically specific lipid bilayers, appropriate acyl chain deuteration allows the vibrational dynamics of each chain moiety to be monitored separately. We have continued the utilization of both Raman and infrared spectroscopic techniques, in conjunction with freeze-quenching methodologies, toward examining the bilayer series comprised of 1-eicosanoyl(d39)-2-eicosenoyl-sn-glycero-3-phosphocholine [C(20-d39):C(20:1 delta j)PC, with j=5, 8, 11, and 13]. The acyl chain packing characteristics and microdomain formation of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC-d31) bilayers were also investigated, Established order/disorder parameters pertinent to each lipid class and to each chain system were determined. Polycrystalline samples were examined using Raman spectroscopic techniques, while infrared spectroscopic procedures were applied to the aqueous bilayer dispersions. Computational techniques provided estimates of chain cluster sizes, which vary between 3-19 saturated acyl chains for these various series of phospholipids. For example, the largest sn-1 lipid chain domain, originating specifically from van der Waals interactions between the chains of neighboring molecules, occurs for the C(20-d39):C(20:1 delta 5)PC species. Using these spectroscopic approaches, we have also examined in detail the microheterogeneity of a variety of bilayer assemblies comprised of highly unsaturated and completely saturated lipid assemblies. (1B) Membrane complexes of sphingolipid and cholesterol, or rafts, have been recently proposed as having roles involving cell trafficking, signal transduction and a variety of membrane related activities. In order to assess the structural properties and domain behavior of these putative complexes, infrared spectroscopic techniques were used to investigate the interactions of binary and ternary mixtures comprised of non-hydroxy fatty acid galactocerebroside (GalCer) and cholesterol in the presence and absence of membrane bilayer matrices of dipalmitoylphosphatidylcholine. In the lipid gel phase, cholesterol acts preferentially with the interfacial region of GalCer. Additionally, cholesterol induces the longer GalCer chains to pack into orthorhombic subcell clusters supporting the existence of domains in the sphingolipid and cholesterol-rich ordered phase in the plasma membrane. (2) Emphasis has been placed on enhancing our mid-infrared spectroscopic chemical imaging microscopy techniques by combining step-scan interferometry with state-of-the-art infrared senstive two-dimensional focal plane array detectors. The integration of high performance digital imaging with noninvasive, high resolution optical spectroscopy allows a visualization of the spatial distribution of distinct chemical species in a variety of host environments. The power of the technique is also manifest in the simultaneous acquisition of an infrared spectrum for each spatial location. This approach has also been extended to the visible spectral region in which reflectance spectra are obtained using a CCD detector and appropriate liquid crystal tunable filters for wavelength discrimination. This particular reflectance unit has been used clinically in assessing blood flow in the skin of burn victims. As examples of the utility of the technique in diagnostic pathology, promising results were obtained from prostate tissue sections and sections from a particular skin lesion (Birt-Hogg-Dube syndrome). The infrared absorbance and Raman emission images, along with their associated vibrational spectra, provided qualitative descriptions of the biochemical differences between unstained tissue from diseased and control animals. Statistical analyses of the individual spectra reflecting the various cell types comprising the tissue provided concise quantitative descriptions of the observed biochemical variations.