Spectroscopy of Biomembrane Monolayers

描述(申请人提供)：肺表面活性物质是哺乳动物肺泡的一个组成部分，用于降低表面张力和促进肺内的氧气摄取。缺乏表面活性物质会导致婴儿和成人的呼吸窘迫综合征。在体内，表面活性物质含有四种脱辅基蛋白，其中两种(SP-B和SP-C)与这种材料的表面活性有关。SP-B被证明可以增强吸附，而SP-C脂蛋白的α-螺旋形式被证明能最大限度地疏水与脂类相互作用，以增加单层的机械稳定性，并催化在空气-水界面形成三维脂蛋白储存库。此外，最近的研究表明，SP-C的脱酰化形式是亚稳定的，并经历了α-螺旋到β-折叠的重排，导致了外观上类似淀粉样纤维的蛋白质聚集体。在肺泡蛋白沉积症患者的支气管肺泡液中发现了这些纤维，主要由脱酰的SP-C组成。分离表面活性物质的特定脂类和蛋白质组分的进展已经发展到可以利用生物物理方法来研究特定结构-功能关系的地步。我们将红外光谱和光学显微镜应用于二维薄膜中表面活性剂模型体系的研究。我们的结果为疏水表面活性蛋白形成表面相关的3-D颗粒提供了直接证据。使用一种新的计算方法，我们已经确定了SP-B和SP-C中以前未知的结构中间体，并跟踪了它们的重定向。此外，我们发现pH是影响去酰化SP-C形成淀粉样蛋白纤维的机制之一。在下一个赠款期间，我们计划将这些类型的测量扩展到模拟生理相关的肺表面活性物质模型的生物物理单分子层。结合突变、振动光谱和光学显微镜，我们将研究SP-B影响单层表面活性的特殊结构机制以及脱酰化SP-C形成淀粉样纤维的机制。

DESCRIPTION (provided by applicant): Pulmonary surfactant is an integral component of mammalian alveoli that serves to reduce surface tension and promote oxygen uptake in the lung. The lack of surfactant leads to respiratory distress syndrome in both infants and adults. In-vivo, surfactant contains four apoproteins, two of which (SP-B and SP-C) have been implicated in the surface-active properties of this material. SP-B has been shown to enhance adsorption, while the alpha-helix form of the SP-C lipoprotein has been shown to maximize hydrophobic interactions with lipids in order to increase the mechanical stability of the monolayer, and catalyze the formation of 3-D lipid-protein reservoirs at the air-water interface. Also, recent research has demonstrated that the deacylated form of SP-C is metastable and undergoes an alpha-helix to beta-sheet rearrangement resulting in protein aggregates that visually resemble amyloid fibrils. These fibrils are found in broncho-alveolar fluid isolated from patients with pulmonary alveolar proteinosis, and consist largely of deacylated SP-C. Progress in isolating specific lipid and protein components of surfactant has advanced to the point where biophysical approaches may be utilized to study specific structure-function relationships. We have applied IR spectroscopy as well as optical microscopy to the study of surfactant model systems in 2-D films. Our results have demonstrated direct evidence for the formation of surface-associated 3-D particles by hydrophobic surfactant proteins. Using a new computational approach, we have identified previously unknown structural intermediates in SP-B and SP-C and followed their reorientation. Furthermore, we have identified pH as one of the mechanisms that influence the formation of amyloid fibrils by deacylated SP-C. In the next grant period, we plan to expand these types of measurements to biophysical monolayers that mimic physiologically relevant pulmonary surfactant models. Using a combination of mutagenesis, vibrational spectroscopy and optical microscopy, we will examine the particular structural mechanisms by which SP-B influences monolayer surface activity as well as the mechanisms by which deacylated SP-C forms amyloid fibrils.