Genetic Optimization of Membrane Protein Production for Crystallization

描述（由申请人提供）：跨膜蛋白（TMP）具有重要的生理作用，是大多数临床有用药物的靶点，占大多数基因组的20-30%，但它们在结构水平上的特征仍然很差。高分辨率结构仅可用于10个不相关的真核TMP（不包括线粒体和叶绿体蛋白）。确定真核TMP结构的两个主要障碍是难以实现天然构象中TMP的高水平表达和TMP的明显不稳定性，导致在表达、纯化和X射线衍射结晶期间天然构象的异质性和损失。本申请描述了一组用于克服这些障碍的遗传方法，其集中于酵母作为遗传上易处理的生物体，其是具有用于X射线晶体学的真核TMP的最成功生产历史的表达宿主。总体方法将是使用流式细胞术结合特异性结合TMP天然构象的荧光配体来筛选突变的和以其他方式修饰的酵母细胞的大型文库，以鉴定增强TMP表达水平和稳定性的改变。这些程序将首先应用于G蛋白偶联受体（GPCR），包括哺乳动物GPCR，可以在酵母和内源性酵母受体1-交配信息素表达。该项目开发的程序和高表达菌株预计都适用于其他类别的TMP。基于荧光配体结合的筛选将用于：1）鉴定酵母宿主菌株中导致功能性受体表达增加的改变。其中一些改变将使用一种新的方法引入，即全球转录机器工程，该工程提供了同时改变多个基因表达的能力。其他改变将使用来源于酵母基因组缺失收集的菌株和通过用过表达的酵母基因和哺乳动物cDNA文库转化受体表达菌株来实现; 2）鉴定编码GPCR的基因中导致表达增加的突变;和3）鉴定使GPCR对热变性具有抗性的突变。由于实现了在酵母中表达的稳定化GPCR的高水平表达，因此将纯化受体，测试其在洗涤剂溶液中的稳定性，并进行结晶试验以确定结构。公共卫生相关性：该项目的重点是开发遗传方法，以帮助确定跨膜蛋白的结构。鉴于真核生物膜蛋白的已知结构数量很少，并且它们具有不同的医学相关性，提供解决甚至一些这样的结构的方法可能对理解这些蛋白质的功能以及设计药物以改变它们的行为产生很大影响。这些新方法将首先应用于G蛋白偶联受体的结构测定，这些受体是大部分临床有用药物的靶点，并在各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.