Overcoming the Barriers to Structural Analysis of GPCRs

描述（由申请人提供）：迫切需要G蛋白偶联受体的额外高分辨率结构信息。初步数据来自C. Sanders，T. Iverson和R. Breyer的研究表明，许多人A类G蛋白偶联受体（GPCR）可以异源过表达并以足以用于结构生物学的量纯化，并且可以获得受体的晶体和溶液NMR光谱。然而，从我们的初步数据中可以清楚地看出，需要新的工具和方法来改善样品条件，以获得功能齐全的受体并提高结构数据的质量。该提案的目的是致力于开发必要的新技术。重视开发廉价和易于使用的方法，以便尽可能多的实验室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，并与现有的最佳技术结合起来。我们还专注于在X射线晶体学，NMR光谱学和GPCR结构和机制的其他生物物理方法中同样有用的方法。具体目标1。开发用替代结构元件替换大多数A类GPCR中发现的关键天然二硫键的方法。这将允许在外源表达后产生功能性受体，而不必首先实现通常非常困难的天然二硫键配对。具体目标2。测试新型表面活性剂，其设计用于在胶束和双胶束条件下稳定GPCR，并模拟天然膜的功能基本组分。双胞形成双极表面活性剂将作为增强受体稳定性的途径进行测试。我们还将测试新的胶束相容的胆固醇衍生物，以满足胆固醇在天然条件下对许多受体发挥的功能性重要作用。具体目标3。开发基于模拟自然界采用的迭代方法的GPCR重折叠方法，以实现真核细胞膜蛋白的折叠。特别是，我们将开发用于GPCR的人工折叠分子伴侣。这些目标将伴随着溶液NMR和结晶筛选，以便在开发新方法时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.