LIPID CONTROL OF MEMBRANE PROTEIN ORGANIZATION

这项工作的长期目标是确定 脂质和蛋白质在相互作用时发生变化， 这些变化与膜的生化功能有关 蛋白质，以及肺表面活性物质的生理功能。 从具体实例中提取的原则加强了 了解生物膜的组织结构， 组织在病理状态下可能会改变。 将追求三个具体目标： (1)为了在生物学上确定精确的酰基链旋转异构体群体， 相关的、构象无序的磷脂相。 为了实现目标 1，两种新的傅里叶变换红外（FT-IR）方法将被应用于 酰基链反式- 用于评价特定的无序形式 （扭结、双弯等）。 (2)为了确定主要膜成分的存在如何改变细胞膜的结构， 磷脂的构象状态分布。 到 实现目标2，FT-IR方法将应用于重构二元 复杂性增加的系统（磷脂/胆固醇或 磷脂/CaATP酶），最后是完整的生物体（A. laidlawii） 其中质膜中的链长可以被控制。 (3)利用在目标1和2中获得的结构性见解， 结构/功能关系在生理上是必要的，但 生物药理学上仍然可控的系统，肺表面活性剂，将在 体外 主要的表面活性蛋白将被分离和重组 加入适当的脂质混合物。 蛋白质二级结构和 将监测薄脂质膜中有序链段的取向 偏振衰减全反射FT-IR。磷脂单层膜 天然表面活性剂和重构体系中结构将 用FT-IR外反射新技术监测 在空气-水界面处的原位光谱。 实验将 直接测试表面活性剂功能的“挤出”假设，即 DPPC，主要的脂质成分，在表面富集， 连续的压缩循环，以产生所需的零 表面张力

The long term objective of this work is to determine how the structures of lipids and proteins are altered upon their mutual interaction, and how these alterations are related to the biochemical functions of membrane proteins, and to the physiological functions of lung surfactant. Principles extracted from specific examples strengthen the basis for understanding the organization of biological membranes and how this organization may be altered during pathological states. Three specific aims will be pursued: (1) To determine precise acyl chain rotamer population in biologically relevant, conformationally disordered phospholipid phases. To achieve Aim 1, two new Fourier-transform infrared (FT-IR) methods will be applied for the quantitative, position-dependent determination of acyl chain trans- gauche isomerization and for the evaluation of specific disordered forms (kinks, double gauche, etc.). (2) To determine how the presence of major membrane components alters the distribution of conformational states available to phospholipids. To achieve Aim 2, the FT-IR approaches will be applied to reconstituted binary systems of increasing complexity (phospholipid/cholesterol or phospholipid/CaATPase) and finally to an intact organism (A. laidlawii) where the chain lengths in the plasma membrane can be controlled. (3) To use the structural insights gained in Aims 1 and 2 for evaluation of structure/function relationships in a physiologically essential, yet biophysically still manageable system, lung surfactant, to be studied in vitro. The major surfactant proteins will be isolated and reconstituted into appropriate lipid mixtures. Protein secondary structure and orientation of the ordered segments in thin lipid films will be monitored with polarized attenuated total reflectance FT-IR. Phospholipid monolayer structure in native surfactant and in reconstituted systems will be monitored with the novel technique of FT-IR external reflection spectroscopy in situ at the air-water interface. The experiments will directly test the "squeezing-out" hypothesis of surfactant function, namely that DPPC, the main lipid component, becomes enriched at the surface during successive compression cycles, in order to produce the requisite zero surface tension.