Overcoming the Barriers to Structural Analysis of GPCRs

描述(由申请人提供)：生物医学上迫切需要关于G蛋白偶联受体的额外的高分辨率结构信息。来自C.Sanders，T.Iverson和R.Breyer实验室的初步数据表明，许多人类A类G蛋白偶联受体(GPCRs)可以异源过度表达和纯化，其数量足以满足结构生物学的要求，并且有可能获得这些受体的晶体和溶液核磁共振谱。然而，从我们的初步数据可以清楚地看出，需要新的工具和方法来改善样本条件，以获得完全功能的受体，并提高结构数据的质量。这项提议的目的是致力于开发必要的新技术。重点是开发廉价和易于使用的方法，以使尽可能多的实验室能够最大限度地利用这些方法。新的方法也被设计成模块化的，这样它们就可以很容易地相互结合，并与现有技术的最佳结合。我们还将重点介绍在X射线结晶学、核磁共振光谱学和其他生物物理方法中同样有用的方法，以了解GPCRs的结构和机制。具体目标1.开发用替代结构元素取代大多数A类GPCRs中的关键天然二硫键的方法。这将允许功能受体在外源表达后产生，而不必首先实现天然的二硫键配对，这通常是非常困难的。具体目标2.测试新型表面活性剂，该表面活性剂既可在胶束和双胶束条件下稳定GPCRs，又可模拟天然膜的功能必需成分。形成双电池的双极表面活性剂将作为增强受体稳定性的一种途径进行测试。我们还将测试新的胶束相容的胆固醇衍生物，以确定它们在天然条件下履行胆固醇对许多受体所起的功能关键作用的能力。具体目标3.开发基于模仿自然界为实现真核膜蛋白折叠而采取的迭代方法的GPCRs重折叠方法。特别是，我们将开发用于GPCR的人工折叠伴侣。这些目标将伴随着溶液核磁共振和结晶筛选，以便随着新方法的发展直接评估结构生物学结果。

DESCRIPTION (provided by applicant): There is a compelling biomedical need for additional high resolution structural information on G protein-coupled receptors. Preliminary data is presented from collaboration between the labs of C. Sanders, T. Iverson, and R. Breyer showing that many human Class A G-protein coupled receptors (GPCRs) can be heterologously overexpressed and purified in quantities sufficient for structural biology, and that it is possible to obtain both crystals and solution NMR spectra of the receptors. However, it is clear from our preliminary data that novel tools and approaches are required to improve sample conditions so as to attain fully functional receptor and to enhance the quality of the structural data. The aims of this proposal are dedicated to developing the requisite novel technology. A premium is placed on developing methods that are inexpensive and easy to use, so as to maximize their accessibility by as many labs as possible. The new approaches are also designed to be modular, so that they can be readily integrated with each other and with the best of existing technology. We also focus on approaches that will be equally useful in X-ray crystallography, NMR spectroscopy, and other biophysical approaches to GPCR structure and mechanism. Specific Aim 1. Develop methods to replace the critical native disulfide bond found in most Class A GPCRs with alternative structural elements. This will allow functional receptor to be generated following exogenous expression without first having to achieve native disulfide pairing, which is often very difficult. Specific Aim 2. Test novel surfactants that are designed both to stabilize GPCRs under micellar and bicellar conditions, and to mimic functionally-essential components of native membranes. Bicelle-forming bipolar surfactants will be tested as a route to enhancing receptor stability. We will also test new micelle-compatible cholesterol derivatives for their ability to fulfill the functionally-essential role that cholesterol plays for many receptors under native conditions. Specific Aim 3. Develop methods for refolding GPCRs that are based on mimicking the iterative approach taken by nature to achieve folding of eukaryotic membrane proteins. In particular, we will develop artificial folding chaperones for GPCRs. These aims will be accompanied by solution NMR and crystallization screens, so as to directly evaluate structural biological outcomes as new methods are developed.