Multi-Level Optimization of Membrane Proteins for Crystallography

跨膜蛋白(TMP)占大多数的蛋白质编码潜力的25%以上。 基因组。它们在细胞和生物体生理学中也发挥着核心作用，并且是一种 所有临床上有用的药物的一部分。然而，我们对这一结构的了解存在着巨大的缺陷 以及TMPS的功能与可溶性蛋白的比较。这主要可以归因于 在将X射线结晶学应用于TMP时通常会遇到很大的障碍。这 应用程序将分子生物学中具有不同背景的三个独立PI聚集在一起， 生物化学、生物物理学和结构生物学，特别是膜蛋白的结构生物学，以创建一条管道 用于TMP结构的测定。这条管道的一个核心原则是需要尽早进行歧视 在可进行结构测定的蛋白质靶标和蛋白质靶标之间可能的生产阶段 那些不是的。考虑到这一点，我们建议以直系、并列和 突变蛋白，携带多个变异体，通过早期阶段的纯化和鉴定 以便最大限度地将目标家庭中最容易驯服的成员提升到关键位置 成功的结晶和衍射物。该项目利用了现有的克隆和表达 最适合平行表达的细菌和酵母表达系统的方案 策略，但将使用杆状病毒表达一些蛋白质。将进行表情测试 使用小规模种植；将以中等规模生产多种形式的给定目标，以 允许使用现有的高通量筛选进行表征，以实现洗涤剂兼容性、生物物理 以及生化表征和小规模探索性结晶试验。只有最多的 有希望的候选人将从中等规模的分析带入大规模生产 使用Hauptman-Woodward研究所的设施进行高通量结晶筛选。 根据需要，将使用脂类立方相进行额外的筛查。除了蛋白质之外 预计PSI网络的初步结构确定工作将针对两类蛋白质： 某些涉及脂质合成和脂质的跨膜转运蛋白和酶 附着在蛋白质上。该项目还寻求开发特定荧光的新方法。 未纯化蛋白质的标记和生物物理方法的改进 蛋白质洗涤剂复合体的表征和筛选。

Transmembrane proteins (TMPs) comprise more than 25% of the protein-coding potential of most genomes. They also play central roles in cell and organismal physiology and are the targets of a large fraction of all clinically useful drugs. However, there is a huge deficit in our knowledge of the structure and function of TMPs in comparison to soluble proteins. This can be primarily attributed to the substantial roadblocks generally encountered in applying x-ray crystallography to TMPs. This application brings together three independent PIs with diverse backgrounds in the molecular biology, biochemistry, biophysics, and structural biology, specifically of membrane proteins, to create a pipeline for TMP structure determination. A central tenet of the pipeline is the need to discriminate at the earliest possible stage in production between protein targets that are amenable to structure determination and those that are not. With this in mind, we propose to target families of orthologous, paralogous, and mutated proteins, carrying multiple variants through the early stages of purification and characterization so as to maximize the chances of advancing the most tractable members of a target family to the point of successful crystallization and diffraction. The project makes use of existing cloning and expression protocols for the bacterial and yeast expression systems that are most amenable to parallel expression strategies, but will use bacculovirus expression for some proteins. Expression testing will be conducted using small-scale growths; multiple forms of a given target will be produced at an intermediate scale to allow characterization using an existing high-throughput screen for detergent compatibility, biophysical and biochemical characterization and small-scale exploratory crystallization trials. Only the most promising candidates from intermediate scale analysis will be carried forward to large scale production for high-throughput crystallization screening using the facilities of the Hauptman-Woodward Institute. Additional screening using lipidic cubic phases will be conducted as needed. In addition to proteins expected from the PSI Network, initial structure determination efforts will target two classes of proteins: certain classes of transmembrane transporters and enzymes involved in lipid synthesis and lipid attachment to proteins. The project also seeks to develop new approaches for specific fluorescent labeling of unpurified proteins, and the development of improved methods for biophysical characterization and screening of protein detergent complexes.