SPECTROSCOPY OF BIOMEMBRANE MONOLAYERS

当前建议的总体目标是采用红外光谱和拉曼光谱以及荧光显微镜相结合的光谱/成像方法来研究肺表面活性物质的生物物理单层模型。需要一种结合的方法，因为没有一种方法能够同时完成结构表征和形态成像，以详细了解单层中的构象和空间特性。振动光谱和荧光成像的结合将使分子性质与表面活性剂单层中的介观（即mum尺寸）结构域形态相关联。我们最近的研究结果表明，表面活性剂蛋白与生物膜界面的相互作用可能选择性地发生在具有不同形态特性的位置，例如结构域边界或特定脂质相的区域。因此，需要研究该体系的分子结构性质，如红外或拉曼光谱所报道的，并结合其荧光可视化的形态结构，以更充分地了解体系组分之间的相互作用。本提案中描述的技术组合将标志着表面活性剂模型中蛋白质和脂质之间分子相互作用研究的重要成就，因为我们将能够将特定的分子光谱特性与表面单层的横向组织联系起来。在下一个资助期内，我们会致力达致以下具体目标：1)通过红外表征表面活性剂蛋白质SP-B和SP-C在单层结构上的界面和疏水相互作用，并通过拉曼光谱确定单层中的蛋白质结构；2)通过荧光显微镜表征SP-B和SP-C对单层形态的影响，并通过散射光显微镜确定这些蛋白质如何产生三维表面排除物质。3)利用红外光谱测量二元混合物的微相分离和结构域形成；4)利用空间分辨拉曼显微镜研究表面活性剂单层膜的横向组织和分子结构。

It is the overall aim of the current proposal to employ a combined spectroscopy/imaging approach using IR and Raman spectroscopy as well as fluorescence microscopy for the study of biophysical monolayer models of pulmonary surfactant. A combined approach is needed, since no one method is able to accomplish both structural characterization and morphological imaging for a detailed understanding of conformational and spatial properties in the monolayer. The combination of vibrational spectroscopy and fluorescence imaging will correlate molecular properties with mesoscopic (i.e. mum size) domain morphology in surfactant monolayers. Our recent results suggest that the interaction of surfactant proteins with biomembrane interfaces may occur selectively at locations with distinct morphological properties, such as at domain boundaries or areas of a certain lipid phase. Therefore, investigation of the molecular structural properties of the system, as reported by IR or Raman, in conjunction with its morphological structure, as visualized by fluorescence, is needed to more fully understand the interaction between the system components. The combination of techniques described in this proposal will mark an important achievement for the investigation of the molecular interaction between proteins and lipids in surfactant models in that we will be able to associate specific molecular spectroscopic properties with lateral organization in surface monolayers. During the next grant period, we will address the following specific aims: 1) to characterize the interfacial and hydrophobic interactions of the surfactant proteins SP-B and SP-C on monolayer structure by IR and to determine protein structure in the monolayer by Raman spectroscopy, 2) to characterize the influence of SP-B and SP-C on monolayer morphology by fluorescence microscopy and to determine how these proteins produce three-dimensional, surface-excluded material as seen using scattered light microscopy, 3) to use IR spectroscopy to measure microphase separation and domain formation in binary mixtures, and 4) to use spatially-resolved Raman microscopy to study lateral organization and molecular structure in surfactant monolayer films.