MOLECULAR DYNAMICS AND VIBRATIONAL CHARACTERISTICS OF MEMBRANE ASSEMBLIES

工作概述：我们的研究工作包括两个主要领域：(1)双层脂质对整体膜蛋白结构重组的调节作用；(2)振动拉曼和红外光谱成像技术的仪器开发和应用。（1A）我们对表征生物膜内波动的脂质微域的影响的兴趣最近集中在由脂质单或多不饱和sn-2链和饱和sn-1链组装组成的双层基质中的簇形成。这些脂质微聚集体的侧向压缩性特性有效地对整体膜蛋白内发生的诱导构象变化施加调节影响。在研究光谱特异性脂质双分子层时，适当的酰基链氘化可以单独监测每个链段的振动动力学。我们继续利用拉曼和红外光谱技术，结合冷冻猝灭方法，研究由1-二十烷醇(d39)-2-二十烷醇-sn-甘油-3-磷酸胆碱组成的双层系列[C(20-d39):C(20:1 δ j)PC, j= 5,8,11和13]。研究了1-棕榈酰-2-油基- cn -甘油-3-磷酸胆碱（POPC-d31）双分子层的酰基链填充特性和微结构域形成，确定了与每种脂类和每种链系统相关的有序/无序参数。使用拉曼光谱技术检测多晶样品，而红外光谱程序应用于水双分子层分散体。计算技术提供了链簇大小的估计，这些不同系列的磷脂在3-19个饱和酰基链之间变化。例如，在C(20-d39):C(20:1 δ 5)PC物种中，最大的sn-1脂质链结构域特别起源于相邻分子链之间的范德华作用。利用这些光谱方法，我们还详细研究了由高度不饱和和完全饱和的脂质组件组成的各种双层组件的微观异质性。（1B）鞘脂和胆固醇的膜复合物，或筏，最近被认为参与细胞运输，信号转导和各种膜相关活动。为了评估这些推测的配合物的结构性质和结构域行为，利用红外光谱技术研究了由非羟基脂肪酸半乳糖脑苷（GalCer）和胆固醇组成的二元和三元混合物在双棕榈酰磷脂酰胆碱膜双层基质存在和不存在的情况下的相互作用。在脂质凝胶期，胆固醇优先作用于GalCer的界面区域。此外，胆固醇诱导较长的GalCer链打包成正交的亚细胞簇，支持鞘脂和质膜中富含胆固醇的有序期结构域的存在。(2)通过将步进扫描干涉测量技术与最先进的红外敏感二维焦平面阵列探测器相结合，重点加强了我们的中红外光谱化学成像显微镜技术。高性能数字成像与无创、高分辨率光谱学的集成允许在各种宿主环境中可视化不同化学物质的空间分布。该技术的力量还体现在同时获取每个空间位置的红外光谱。这种方法也被扩展到可见光光谱区域，利用CCD探测器和适当的液晶可调滤波器进行波长辨别，获得反射光谱。这种特殊的反射单位已在临床上用于评估烧伤患者皮肤中的血流。作为该技术在诊断病理学中的应用实例，从前列腺组织切片和特定皮肤病变切片（Birt-Hogg-Dube综合征）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，提供了对患病动物和对照动物未染色组织之间生化差异的定性描述。个体光谱的统计分析反映了组成组织的各种细胞类型，为观察到的生化变化提供了简明的定量描述。

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.