Membrane Protein Structures in Phospholipid Bilayers by Solid State NMR (RMI)

描述(申请人提供)：膜蛋白负责许多基本的生物功能，包括作为最大和最重要的一类药物受体。人类基因组中编码的蛋白质中有20%以上是螺旋膜蛋白，许多人类疾病都是由这些蛋白质的突变引起的。膜蛋白的结构将为了解其功能和设计新药提供必要的信息。然而，这类蛋白质的结构测定对于现有的方法来说是有问题的，迫切需要开发新的方法。 总体目标是开发一种普遍适用的方法来确定磷脂双层中螺旋膜蛋白的三维结构。近期的目标是开发对齐样品的固态核磁共振，并应用这种方法来确定具有两个、三个和四个跨膜螺旋的汞转运膜蛋白家族的结构。这项研究将在最初的例子之间架起桥梁，其中一个跨膜螺旋的结构已经用这种方法确定，而更大的更复杂的蛋白质具有多个跨膜螺旋。最终目标是将这项技术应用于具有七个跨膜螺旋的G蛋白偶联受体(GPCRs)。这项跨学科的研究涉及蛋白质表达、同位素标记和纯化；高排列磷脂双层样品的制备；实验核磁共振光谱；以及蛋白质结构的计算。排列样品的固态核磁共振是一种很有前途的方法，原因如下：1)在生理条件下，这些蛋白质以完全水合的磷脂双层形式存在。2.)固体核磁共振光谱学没有基本的尺寸限制，拟议的研究为未来应用于更大的膜蛋白奠定了基础。3.)所有的主链和侧链共振都可以在通过表达获得的蛋白质中被解析和指认。4.)测量的取向约束产生了具有原子分辨率的结构。5.)可以获得蛋白质结构和动力学的综合视图。

DESCRIPTION (provided by applicant): Membrane proteins are responsible for many essential biological functions, including as the largest and most important class of drug receptors. More than 20% of the proteins encoded in the human genome are helical membrane proteins, and many human diseases result from mutations in these proteins. The structures of membrane proteins will provide information essential for understanding their functions and designing new drugs. However, structure determination of this class of proteins is problematic for current methods, and the development of new methods is urgently needed. The overall goal is to develop a generally applicable method for determining the three-dimensional structures of helical membrane proteins in phospholipid bilayers. The immediate goals are to develop solid-state NMR of aligned samples and to apply this approach to determine the structure of a family of mercury transport membrane proteins with two, three, and four trans-membrane helices. This research will bridge between the initial examples with one trans-membrane helix whose structures have been determined with this approach and larger more complex proteins with multiple membrane spanning helices. The ultimate goal is to apply this technology to G-protein coupled receptors (GPCRs) that have seven trans-membrane helices. This interdisciplinary research involves protein expression, isotopic labeling and purification; preparation of highly aligned phospholipid bilayer samples; experimental NMR spectroscopy; and the calculation of protein structures. Solid-state NMR of aligned samples is a promising approach for the following reasons: 1.) The proteins are in fully hydrated phospholipid bilayers under physiological conditions. 2.) There are no fundamental size limitations to solid-state NMR spectroscopy and the proposed studies set the stage for future applications to larger membrane proteins. 3.) All backbone and side chain resonances can be resolved and assigned in proteins obtained by expression. 4.) The measured orientation constraints yield structures with atomic resolution. 5.) An integrated view of protein structure and dynamics can be obtained.