MOLECULAR DYNAMICS AND VIBRATIONAL CHARACTERISTICS OF MEMBRANE ASSEMBLIES

膜组件的分子动力学和振动特性

• 批准号: 6289744
• 负责人: Ira W. Levin
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6289744
• 关键词: Raman spectrometry; biomedical equipment development; biophysics; image processing; infrared spectrometry; interferometry; lipid bilayer membrane; membrane proteins; membrane structure; molecular dynamics; protein biosynthesis; protein structure

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.

工作总结：我们的研究工作包括两个主要领域：（1）双层脂质对膜蛋白结构重组的调节作用，以及（2）振动拉曼和红外光谱成像技术的仪器开发和应用。(1A)我们的兴趣在表征生物膜内的波动脂质微区的影响最近集中在由脂质单或多不饱和的sn-2链和饱和的sn-1链组装体组成的双层基质内的簇形成。这些脂质微聚集体的侧向可压缩性是有效的，在施加调制的影响，诱导的构象变化内发生的整合膜蛋白。在研究光谱特定的脂质双层，适当的酰基链氘代允许每个链部分的振动动力学分别进行监测。我们继续利用拉曼和红外光谱技术，结合冷冻淬灭方法，研究了由1-二十烷酰基（d39）-2-二十烯酰基-sn-甘油基-3-磷酸胆碱[C（20-d39）：C（20：1 delta j）PC，j=5，8，11和13]组成的双层系列。本文还研究了1-棕榈酰-2-油酰-sn-甘油-3-磷酸胆碱（POPC-d31）双层膜的酰基链堆积特性和微区的形成。使用拉曼光谱技术检查多晶样品，而红外光谱程序应用于水性双层分散体。计算技术提供了链簇大小的估计，对于这些不同系列的磷脂，其在3-19个饱和酰基链之间变化。例如，最大的sn-1脂质链结构域，特别是源于相邻分子链之间的货车德瓦尔斯相互作用，出现在C（20-d39）：C（20：1 delta 5）PC种类中。使用这些光谱方法，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，最近已被提出具有涉及细胞运输，信号转导和各种膜相关活动的作用。为了评估这些推定的复合物的结构特性和域的行为，红外光谱技术被用来调查的二元和三元混合物组成的非羟基脂肪酸半乳糖苷（GalCer）和胆固醇的存在和不存在的膜双层基质的二棕榈酰磷脂酰胆碱的相互作用。在脂质凝胶相中，胆固醇优先与GalCer的界面区域起作用。此外，胆固醇诱导较长的GalCer链包装成斜方晶系亚细胞簇，支持质膜中鞘脂和富含胆固醇的有序相中存在结构域。(2)重点放在提高我们的中红外光谱化学成像显微镜技术相结合的步进扫描干涉与国家的最先进的红外敏感的二维焦平面阵列探测器。高性能数字成像与非侵入性、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。该技术的威力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谱区域，其中反射光谱是使用CCD检测器和适当的液晶可调谐滤波器的波长歧视。这种特殊的反射单元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实用性的例子，从前列腺组织切片和特定皮肤病变（Birt-Hogg-Dube综合征）的切片获得了有希望的结果。红外吸收和拉曼发射图像，沿着其相关的振动光谱，提供了来自患病动物和对照动物的未染色组织之间的生化差异的定性描述。对反映组织中各种细胞类型的单个光谱的统计分析提供了对所观察到的生化变化的简明定量描述。