Structure and Function of Lens Membrane Proteins

描述(申请人提供)：人工晶状体是一种独特的结构，设计精巧，可以将光线聚焦到视网膜上，这一过程需要根据眼睛与所聚焦对象的距离来改变晶状体的形状。我们感兴趣的是晶状体中膜蛋白的结构和功能，它们在维持晶状体的动态平衡和透明度方面起着至关重要的作用。膜通道和转运体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。膜蛋白还调节纤维细胞的紧密堆积，从而帮助避免晶状体组织的光散射。这项建议关注晶状体纤维细胞中的两种主要膜蛋白，即Tetraspanin MP20和水通道AQP0。使用二维(2D)AQP0晶体，我们也在努力了解脂类与膜蛋白非特异性相互作用的一般原理。在之前的资助期间，我们已经制作了AQP0的双层2D晶体的1.9？密度图，它解析了蛋白质周围的脂分子。这项建议的具体目标1是进一步研究非特异性脂质-蛋白质相互作用。我们将扩展电子结晶学数据集来模拟AQP0周围脂类的替代构象。我们还将尝试将密度图的分辨率扩展到1.5°，这可能允许我们可视化AQP0中残基的电荷。我们特别感兴趣的是组氨酸44和60在不同pH值下的电荷状态，因为这两个残基参与了AQP0对水传导的pH调节。此外，我们将在2D晶体中可视化AQP0，这些晶体使用具有不同酰基链和头基的脂类生长。这些实验将使我们能够系统地研究定义非特异性脂蛋白相互作用的脂类特性，并了解脂类和蛋白质如何相互适应。我们还获得了AQP0的六方2D晶体，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成四聚体组装成方形阵列的蛋白质如何形成六方2D晶体。关于MP20和其他Tetraspanins的结构信息仍然很稀少。因此，具体目标3主要是通过电子显微镜来确定MP20的结构，但也要进行X射线结晶学研究。获得的MP20的结构信息可能有助于对Tetraspanin家族其他成员的结构进行建模，这些成员在许多细胞过程中都是重要的，如细胞黏附、增殖、激活、迁移和凋亡。在特定的目标4中，我们将通过研究MP20与重要的黏附调节剂Galectin-3的相互作用来表征其作为细胞黏附分子的功能。我们还将确定MP20是否能与晶状体特异性整合素结合，因为已知许多Tetraspanins与整合素相互作用，特别是当这些整合素含有？1亚基的时候。公众健康相关性水通道蛋白-0和MP20对晶状体正常功能的重要性可以从这两种膜蛋白中的任何一种突变导致白内障的形成这一事实来说明。大量的生化和生物物理研究也证实了蛋白质-脂质相互作用对膜蛋白的组装、稳定性和功能的重要性。关于脂类如何影响膜蛋白的结构和功能，人们几乎一无所知，膜蛋白的功能障碍是导致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.