Lipid and Protein Effects on Monolayer Stability

描述(申请人提供)：需要检验的假设是，最佳表面活性剂必须在双层中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炼，以在单层中产生更高的固相分数，以促进足够大的表面粘度，以促进高单层坍塌压力(低最低表面张力)以及将表面活性剂保留在肺泡中。这些相互排斥的要求要求与空气-水界面相邻的表面活性剂“储存库”同时由从溶液中吸附的单层塌陷结构和双层结构供给。理论预测的塌陷结构、界面吸附速率和表面活性剂与界面的粘附性将三维(3D)结构与表面剪切粘度、弹性、弯曲弹性、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。为了检验这些理论并利用它们来确定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邻界面的10-100微米。此外，还将使用表面粘度、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子显微镜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。根据已知的疏水性表面活性蛋白SP-B和SP-C的氨基酸序列，设计并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物质功能的影响。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结构域(例如，两亲性-螺旋序列)。SP-B多肽将基于NK-赖氨酸的模板结构，并将包括单体和二聚体结构。SP-C蛋白构建将基于已知的天然猪SP-C的核磁共振溶液结构和一个重组的全长SP-C，还将包括一个新的二聚体SP-C。这些多肽和突变体将被用来鉴定参与多肽与表面活性脂类的二级结构、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限度地提高体内活性所必需的多肽特性。

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.