Genetic Optimization of Membrane Protein Production for Crystallization

描述（由申请人提供）：跨膜蛋白（TMPs）发挥着重要的生理作用，是大多数临床有用药物的靶点，占大多数基因组的20-30%，然而在结构水平上对它们的表征仍然很差。高分辨率结构仅可用于10个不相关的真核生物tmp（不包括线粒体和叶绿体蛋白质）。确定真核TMPs结构的两个主要障碍是难以以天然构象高水平表达TMPs，以及TMPs在表达、纯化和x射线衍射结晶过程中明显的不稳定性导致天然构象的异质性和损失。本申请描述了一套克服这些障碍的遗传程序，重点关注酵母作为一种遗传易处理的生物，是具有最成功的真核TMPs生产历史的表达宿主，用于x射线晶体学。总体方法将是使用流式细胞术结合荧光配体特异性结合TMPs的天然构象来筛选大量的突变和其他修饰的酵母细胞文库，以确定提高TMPs表达水平和稳定性的改变。这些方法最初将应用于G蛋白偶联受体（gpcr），包括可以在酵母和内源性酵母1-交配信息素受体中表达的哺乳动物gpcr。该项目开发的方法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)确定酵母菌宿主菌株中导致功能受体表达增加的变化。其中一些改变将通过一种新的方法引入，即全球转录机械工程，它提供了同时改变多个基因表达的能力。其他改变将通过酵母基因组缺失收集的菌株和用过表达的酵母基因和哺乳动物cdna文库转化表达受体的菌株来实现；2)鉴定编码gpcr的基因突变导致表达增加；3)鉴定使gpcr抗热变性的突变。随着稳定的gpcr在酵母中的高水平表达的实现，受体将被纯化，在洗涤剂溶液中测试稳定性，并进行结晶试验以确定结构。公共卫生相关性：该项目侧重于开发遗传方法，以帮助确定跨膜蛋白的结构。考虑到真核膜蛋白的已知结构数量很少，以及它们的多种医学相关性，提供解决这些结构的方法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重大影响。新方法将首先应用于G蛋白偶联受体的结构测定，G蛋白偶联受体是大部分临床有用药物的靶点，在多种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.