Genetic Optimization of Membrane Protein Production for Crystallization

描述(申请人提供)：跨膜蛋白(TMP)发挥着重要的生理作用，是大多数临床有用药物的靶标，占大多数基因组的20%-30%，但它们在结构水平上的特征仍然很差。只有10个不相关的真核生物TMP(不包括线粒体和叶绿体蛋白)的高分辨率结构可用。确定真核生物TMPS结构的两个主要障碍是难以在天然构象中实现高水平的表达，以及TMPS在X射线衍射的表达、纯化和结晶过程中明显的不稳定性，导致天然构象的异质性和丢失。本申请描述了一套克服这些障碍的遗传程序，重点放在酵母作为一种遗传易处理的有机体，它是表达宿主，拥有用于X射线结晶学的真核细胞TMPS生产的最成功的历史。总体方法是结合结合天然构象的TMPS的荧光配基，使用流式细胞术来筛选突变和其他修饰的酵母细胞的大型文库，以确定提高TMPS表达水平和稳定性的变化。这些程序最初将应用于G蛋白偶联受体(GPCRs)，包括可在酵母中表达的哺乳动物GPCRs和1-交配信息素的内源酵母受体。该项目开发的程序和高表达菌株有望适用于其他类型的TMP。基于荧光配体结合的筛选将用于：1)鉴定导致功能性受体表达增加的酵母宿主菌株的变化。其中一些改变将使用一种新的方法，即全球转录机械工程，提供同时改变多个基因表达的能力。其他改变将通过使用来自酵母基因组缺失收集的菌株和通过用过表达的酵母基因库和哺乳动物cDNA来转化受体表达的菌株来实现；2)确定编码GPCRs的基因中导致表达增加的突变；以及3)确定使GPCRs抵抗热变性的突变。随着稳定的GPCRs在酵母中得到高水平表达，受体将被提纯，在洗涤剂溶液中进行稳定性测试，并进行结晶试验以确定结构。公共卫生相关性：该项目的重点是开发基因方法，以帮助确定跨膜蛋白的结构。鉴于真核膜蛋白的已知结构数量很少，而且它们的医学相关性很大，提供即使是少数几个这样的结构的方法也可能对理解这些蛋白质的功能和设计改变其行为的药物产生很大影响。这些新方法将首先应用于G蛋白偶联受体的结构测定，G蛋白偶联受体是一大部分临床有用药物的靶标，并在广泛的信号通路中发挥关键作用。

DESCRIPTION (provided by applicant): Transmembrane proteins (TMPs) play important physiological roles, are the targets of most clinically useful drugs, and constitute 20-30% of most genomes, however they remain poorly characterized at the structural level. High-resolution structures are available for only ten unrelated eukaryotic TMPs (excluding mitochondrial and chloroplast proteins.) Two major barriers to determining structures of eukaryotic TMPs are the difficulty of achieving high levels of expression of TMPs in native conformations and the apparent instability of TMPs leading to heterogeneity and loss of native conformations during expression, purification, and crystallization for x-ray diffraction. This application describes a set of genetic procedures for overcoming these barriers focusing on yeast as a genetically tractable organism that is the expression host with the most successful history of production of eukaryotic TMPs for x-ray crystallography. The overall approach will be to use flow cytometry in conjunction with fluorescent ligands that bind specifically to native conformations of TMPs to screen large libraries of mutated and otherwise modified yeast cells to identify alterations that enhance the levels of expression and stabilities of TMPs. These procedures will initially be applied to G protein Coupled Receptors (GPCRs), including mammalian GPCRs that can be expressed in yeast and the endogenous yeast receptor for 1-mating pheromone. Both the procedures and the high-expressing strains developed by the project are expected to be applicable to other classes of TMPs. Screening based on binding of fluorescent ligands will be used to: 1) Identify alterations in yeast host strains leading to increased expression of functional receptors. Some of these alterations will be introduced using a new approach, global transcription machinery engineering that provides the ability to alter expression of multiple genes at the same time. Other alterations will be effected using strains derived from yeast genomic deletion collection and by transforming receptor-expressing strains with libraries of overexpressed yeast genes and mammalian cDNAs; 2) Identify mutations in the genes encoding GPCRs that lead to increased expression; and 3) Identify mutations that render GPCRs resistant to thermal denaturation. As high-level expression of stabilized GPCRs expressed in yeast is achieved, the receptors will be purified, tested for stability in detergent solutions, and subjected to crystallization trials for structure determination. PUBLIC HEALTH RELEVANCE: This project focuses on the development of genetic approaches to aid in determination of the structures of transmembrane proteins. Given the small number of known structures of eukaryotic membrane proteins, and their diverse medical relevance, providing methods to solve even a few such structures could have a large impact on understanding of the functions of such proteins and in the design of drugs to alter their behaviors. The new approaches will be applied initially to structure determination of G protein coupled receptors, which are targets of a large fraction of clinically useful drugs and play critical roles in a wide variety of signaling pathways.