SPECTROSCOPY OF BIOMEMBRANE MONOLAYERS

当前提议的总体目标是使用红外光谱和拉曼光谱以及荧光显微镜的组合光谱/成像方法来研究肺表面活性物质的生物物理单层模型。由于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属性，因此需要一种组合的方法。振动光谱和荧光成像的结合将把分子性质与表面活性剂单分子膜中的介观(即最小尺寸)结构域形态联系起来。我们最近的结果表明，表面活性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征的位置，例如结构域边界或特定脂相的区域。因此，需要研究体系的分子结构性质，如红外光谱或拉曼光谱所报道的那样，结合其形态结构，如荧光所显示的，以更全面地了解体系组分之间的相互作用。这项建议中描述的技术组合将标志着在表面活性剂模型中研究蛋白质和脂类之间的分子相互作用的一项重要成就，因为我们将能够将特定的分子光谱性质与表面单分子层的横向组织联系起来。在下一个授权期内，我们将致力于以下具体目标：1)通过IR表征表面活性蛋白质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，并通过散射光学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中微相分离和结构域的形成；以及4)使用空间分辨拉曼显微镜研究表面活性单层膜中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.