Lipid and Protein Effects on Monolayer Stability

脂质和蛋白质对单层稳定性的影响

• 批准号: 7074637
• 负责人: Joseph Anthony Zasadzinski
• 金额: $ 25.95万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7074637
• 关键词: X ray crystallography; adsorption; atomic force microscopy; biomaterial development /preparation; biomaterial interface interaction; biotechnology; biotherapeutic agent; chemical structure function; cholesterol; combinatorial chemistry; confocal scanning microscopy; elasticity; fluorescent dye /probe; intermolecular interaction; lipid solubility; lipid structure; molecular film; peptide chemical synthesis; phase change; protein folding; protein structure; pulmonary surfactants; respiratory distress syndrome of newborn; surface property; synthetic protein; viscosity

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.

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。然后必须精制该组合物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塌陷压力（低的最小表面张力）以及将表面活性剂保留在泡中。这些相互排斥的要求要求在空气-水界面附近形成表面活性剂“储存库”，该表面活性剂“储存库”由从溶液中吸附的单层塌陷结构和双层结构同时供给。塌陷结构的理论预测、界面的吸附速率以及表面活性剂对界面的粘附力将 3 维 (3D) 结构与包括表面剪切粘度、弹性、弯曲模量、表面压力等在内的参数联系起来。这些参数取决于脂质和蛋白质的组成决定了流体与固体的共存。胆固醇在确定表面粘度和弹性方面也起着重要作用。为了测试这些理论并使用它们来定义替代表面活性剂成分，将开发共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，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用于将表面活性剂单层和双层性质与表面活性剂组合物联系起来。将设计并合成基于疏水性表面活性剂蛋白 SP-B 和 SP-C 的已知氨基酸序列的合成肽家族，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。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，两亲螺旋序列）。 SP-B 肽将基于 NK-溶素的模板结构，包括单体和二聚体结构。 SP-C 蛋白构建体将基于天然猪 SP-C 的已知 NMR 溶液结构和重组全长 SP-C，并且还将包括新的二聚体 SP-C。这些肽和突变体将用于鉴定参与肽与表面活性剂脂质的二级结构、三级折叠和四级缔合的关键氨基酸。这些研究的总体目标是了解最大化体内活性所需的肽特征。