Lipid and Protein Effects on Monolayer Stability

描述（由申请人提供）：要测试的假设是，最佳表面活性剂必须在双层中具有相对较大比例的无序流体相，以促进低溶液粘度和快速吸附到界面。然后，必须对该组合物进行精制，以在单层中产生更高的固相分数，以提高表面粘度，从而提高单层的高崩溃压力（最低表面张力低），并将表面活性剂保留在肺泡中。这些相互排斥的要求要求在空气-水界面附近形成一个表面活性剂“储层”，该储层由从溶液中吸附的单层坍塌结构和双层结构同时提供。崩溃结构、界面吸附速率和表面活性剂与界面的粘附力的理论预测将三维（3D）结构与表面剪切粘度、弹性、弯曲模量、表面压力等参数联系起来。这些参数取决于脂质和蛋白质组成，这决定了流固共存。胆固醇在决定表面粘度和弹性方面也起着重要作用。为了验证这些理论并使用它们来定义替代表面活性剂的组成，将开发共聚焦显微镜来研究表面活性剂界面和紧挨着界面的10 - 100微米。此外，表面粘度、朗缪尔等温线、荧光光学显微镜、原子力显微镜和冷冻断裂电镜将用于将表面活性剂的单层和双层性质与表面活性剂的组成联系起来。以已知疏水表面活性剂SP-B和SP-C的氨基酸序列为基础，设计和合成了一系列合成肽，以表征序列、二级结构和三级折叠对表面活性剂功能的影响。多肽将包括已知与脂质和膜模拟系统相关的蛋白质的功能域（例如，两性螺旋序列）。SP-B肽将基于NK-lysin的模板结构，并将包括单体和二聚体结构。SP-C蛋白的构建将基于已知的天然猪SP-C和重组全长SP-C的NMR溶液结构，并将包括一个新的二聚体SP-C。这些多肽和突变体将用于识别参与多肽与表面活性剂脂质的二级结构、三级折叠和四级结合的关键氨基酸。这些研究的总体目标是了解最大化体内活性所必需的肽特征。

DESCRIPTION (provided by applicant): The hypothesis to be tested is that an optimal surfactant must have a relatively large fraction of disordered, fluid phase in bilayers to promote a low solution viscosity and rapid adsorption to the interface. This composition must then be refined to produce a higher solid phase fraction in monolayers to promote a surface viscosity large enough to promote high monolayer collapse pressures (low minimum surface tensions) as well as retain surfactant in the alveolus. These mutually exclusive requirements require that a surfactant "reservoir" adjacent to the air-water interface form that is fed simultaneously by monolayer collapse structures and bilayer structures adsorbed from solution. Theoretical predictions of collapse structures, the rates of adsorption to the interface, and the adhesion of surfactant to the interface relate the 3-dimensional (3D) structures to parameters including surface shear viscosity, elasticity, bending modulus, surface pressure etc. These parameters depend on lipid and protein composition, which determines fluid-solid coexistence. Cholesterol also plays an important role in determining surface viscosity and elasticity. To test these theories and to use them to define replacement surfactant composition, confocal microscopy will be developed to investigate the surfactant interface and the 10 - 100 microns immediately adjacent to the interface. In addition, surface viscosity, Langmuir isotherms, fluorescence optical microscopy, atomic force microscopy, and freeze-fracture electron microscopy will be used to relate surfactant monolayer and bilayer properties to surfactant composition. A family of synthetic peptides based on the known amino acid sequences of the hydrophobic surfactant proteins SP-B and SP-C will be designed and synthesized to characterize the effects of sequence, secondary structure and tertiary folding on surfactant function. Peptides will include functional domains (e.g., amphipathic-helical sequences) of the protein known to associate with lipids and membrane-mimic systems. SP-B peptides will be based on the templated structure of NK-lysin and will include monomer and dimer constructs. SP-C protein constructs will be based on the known NMR solution structure of native pig SP-C and a recombinant full length SP-C, and will also include a new dimer SP-C. These peptides and mutants will be used to identify key amino acids that participate in the secondary structure, tertiary folding and quaternary associations of the peptides with surfactant lipids. The overall goal of these studies is to understand the peptide features necessary to maximize in vivo activity.