Structure and Function of Lens Membrane Proteins

描述（申请人提供）：眼透镜是一种独特的结构，其设计精巧，可将光线聚焦到视网膜上，这一过程需要根据眼睛与其聚焦对象的距离使透镜发生实质性的形状变化。我们对透镜中的膜蛋白的结构和功能感兴趣，这些蛋白在维持透镜内环境稳定和透明度方面起着至关重要的作用。膜通道和转运蛋白是微循环系统的基础，微循环系统为深层纤维细胞提供营养，并清除它们的废物。膜蛋白也介导纤维细胞的紧密包装，从而有助于避免透镜组织的光散射。该建议集中于透镜纤维细胞中的两种主要膜蛋白，四跨膜蛋白MP20和水通道AQP 0。使用二维（2D）AQP 0晶体，我们也在努力了解脂质与膜蛋白非特异性相互作用的一般原理。在上一个资助期间，我们已经制作了AQP 0双层2D晶体的1.9 <$D密度图，它解决了蛋白质周围的脂质分子。本提案的具体目标1是进一步研究非特异性脂质-蛋白质相互作用。我们将扩大电子晶体学数据集模型替代构象的脂质周围AQP 0。我们还将尝试将密度图的分辨率扩展到1.5英寸，这可能使我们能够可视化AQP 0中残基的电荷。我们特别感兴趣的组氨酸44和60在不同的pH值的电荷状态，因为这两个残基已经牵连在水传导的pH调节水通过AQP 0。此外，我们将在使用具有不同酰基链和头部基团的脂质生长的2D晶体中可视化AQP 0。这些实验将使我们能够系统地研究定义非特异性脂质-蛋白质相互作用的脂质特征，并了解脂质和蛋白质如何相互适应。我们还获得了AQP 0的六边形2D晶体，并且本提案的具体目标2是使用各种结构和生物物理方法来阐明设计用于形成组装成正方形阵列的四聚体的蛋白质如何形成六边形2D晶体。关于MP20和其他四跨膜蛋白的结构信息仍然很少。因此，具体目标3是主要通过电子显微镜确定MP20的结构，但也追求X射线晶体学。获得的MP20的结构信息可能是有用的模型四跨膜蛋白家族，这是重要的许多细胞过程中，如细胞粘附，增殖，活化，迁移和凋亡的其他成员的结构。在具体目标4中，我们将通过研究MP20与半乳糖凝集素-3（一种突出的粘附调节剂）的相互作用来表征MP20作为细胞粘附分子的功能。我们还将确定MP20是否可以结合到晶状体特异性整合素，因为已知许多四跨膜蛋白与整合素相互作用，特别是如果这些包含一个？1个亚基。水通道蛋白-0和MP20对于正常透镜功能的重要性通过这两种膜蛋白中任一种的突变导致白内障形成的事实来说明。大量的生物化学和生物物理学研究也已经确立了蛋白质-脂质相互作用对于膜蛋白的组装、稳定性和功能的重要性。关于脂质如何影响膜蛋白的结构和功能几乎一无所知，膜蛋白的功能障碍是大量人类疾病的原因。

DESCRIPTION (provided by applicant): The ocular lens is a unique structure exquisitely designed to focus light onto the retina, a process that requires substantial shape changes of the lens according to the distance of the eye from the object it is focusing on. We are interested in the structure and function of membrane proteins in the lens, which play crucial roles in maintaining lens homeostasis and transparency. Membrane channels and transporters are the basis for a microcirculation system that supplies deeper-lying fiber cells with nutrients and clears them of waste products. Membrane proteins also mediate the tight packing of the fiber cells, thus helping to avoid light scattering by the lens tissue. This proposal focuses on the two major membrane proteins in lens fiber cells, the tetraspanin MP20 and the water channel AQP0. Using two-dimensional (2D) AQP0 crystals, we are also working towards understanding the general principles that underlie non-specific interactions of lipids with membrane proteins. In the previous funding period, we have produced a 1.9 ¿ density map of double-layered 2D crystals of AQP0, which resolved the lipid molecules surrounding the protein. Specific Aim 1 of this proposal is to further study non-specific lipid-protein interactions. We will expand the electron crystallographic data set to model alternative conformations of the lipids surrounding AQP0. We will also attempt to extend the resolution of the density map to 1.5 ¿, which may allow us to visualize charges of residues in AQP0. We are particularly interested in the charge state of histidines 44 and 60 at different pH values, as these two residues have been implicated in the pH regulation of water conduction by AQP0. In addition, we will visualize AQP0 in 2D crystals grown using lipids with different acyl chains and head groups. These experiments will allow us to systematically investigate the lipid characteristics that define non-specific lipid-protein interactions and to understand how lipids and proteins adapt to each other. We also obtained hexagonal 2D crystals of AQP0, and Specific Aim 2 of this proposal is to use a variety of structural and biophysical methods to elucidate how a protein designed to form tetramers assembling into square arrays can form hexagonal 2D crystals. Structural information on MP20 as well as other tetraspanins is still sparse. Specific Aim 3 is thus to determine the structure of MP20 primarily by electron microscopy but also pursuing X-ray crystallography. The structural information obtained for MP20 may be useful to model the structure of other members of the tetraspanin family, which are important in many cellular processes, such as cell adhesion, proliferation, activation, migration, and apoptosis. In Specific Aim 4 we will characterize the function of MP20 as a cell adhesion molecule by studying its interaction with galectin-3, a prominent adhesion modulator. We will also determine whether MP20 can bind to lens-specific integrins, as many tetraspanins are known to interact with integrins, especially if these contain a ?1 subunit. PUBLIC HEALTH RELEVANCE The importance of aquaporin-0 and MP20 for proper lens function is illustrated by the fact that mutations in either one of these two membrane proteins lead to the formation of cataracts. A wealth of biochemical and biophysical studies has also established the importance of protein-lipid interactions for the assembly, stability, and function of membrane proteins. Almost nothing is known about how lipids affect the structure and function of membrane proteins, whose dysfunction are the cause of a large number of human disorders.