Genetic Optimization of Membrane Protein Production for Crystallization

跨膜蛋白(TMPS)具有重要的生理作用，是细胞外信号转导的靶点 大多数临床有用的药物，占大多数基因组的20%-30%，但它们仍然 在结构层面上表现得很差。高分辨率结构仅适用于 10个不相关的真核生物TMPS(不包括线粒体和叶绿体蛋白)二 确定真核生物TMPS结构的主要障碍是难以实现 TMPS在天然构象中的高水平表达以及TMPS的明显不稳定性 TMP导致表达过程中天然构象的异质性和丢失， 提纯、结晶，用于X-射线衍射。此应用程序描述了一组 克服这些障碍的遗传程序，重点放在酵母作为一种基因 易驯服的有机体，是生产历史上最成功的表达宿主 用于X-射线结晶学的真核生物TMPS。总体方法将是使用流 结合天然荧光配体的流式细胞术 用TMPS构象筛选大量突变和修饰酵母文库 以确定提高TMPS表达水平和稳定性的变化。 这些程序将首先应用于G蛋白偶联受体(GPCRs)， 包括可在酵母和内源酵母中表达的哺乳动物GPCRs 交配信息素受体。程序菌株和高表达菌株 由该项目开发的TMPS有望适用于其他类别的TMPS。筛选 基于荧光配体的结合将被用于：1)识别酵母中的变化 宿主菌株导致功能性受体表达增加。其中一些 将使用一种新的方法--全局转录机制来引入更改 工程学，提供同时改变多个基因表达的能力。 其他的改变将使用来自酵母基因组缺失的菌株来实现 利用高表达文库收集和转化受体表达菌株 酵母基因和哺乳动物的cDNA；2)确定编码基因的突变 导致表达增加的GPCRs；以及3)识别导致 耐热变性的GPCRs。作为稳定GPCRs的高水平表达 在酵母中实现表达，将对受体进行纯化，测试其在洗涤剂中的稳定性 溶液，并进行结晶试验以确定结构。

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 ¿-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.