LIPID CONTROL OF MEMBRANE PROTEIN ORGANIZATION

这项工作的长期目标是确定 脂类和蛋白质在相互作用时会发生改变，以及它们是如何改变的 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。 从具体例子中提取的原则加强了 了解生物膜的组织结构以及如何 在病理状态下，组织可能会改变。 我们会追求三个具体目标： (1)精确测定生物中的酰链旋转异构体种群 相关的、构象紊乱的磷脂相。为了达到目的 1、将应用两种新的傅里叶变换红外(FT-IR)方法 反式酰基链的位置依赖定量测定 Guche异构化及其对特定无序形式的评价 (扭结、双重笨拙等)。 (2)确定主要膜组分的存在如何改变 磷脂可利用的构象状态分布。至 为了实现目标2，FT-IR方法将应用于重组的二元组 日益复杂的系统(磷脂/胆固醇或 磷脂/CaATPase)，最后是一个完整的生物体(A.laidlawii) 其中质膜中的链长可以控制。 (3)利用目标1和目标2中获得的结构性见解来评估 生理上必不可少的结构/功能关系，但 生物物理上仍可管理的系统，肺表面活性物质，有待研究 体外培养。主要的表面活性蛋白将被分离和重组 变成合适的脂类混合物。蛋白质的二级结构和 将监测薄膜中有序链段的取向 具有偏振衰减全反射FT-IR。磷脂单分子层 天然表面活性剂和重组体系中的结构将 用FT-IR外反射新技术进行监测 空气-水界面的原位光谱。这些实验将会 直接检验表面活性物质作用的“挤出”假说，即 主要的脂质成分DPPC在整个过程中在表面变得丰富 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.