Multi-Level Optimization of Membrane Proteins for Crystallography

用于晶体学的膜蛋白的多级优化

• 批准号: 8152227
• 负责人: MARK E. DUMONT
• 金额: $ 122.58万
• 依托单位: HAUPTMAN-WOODWARD MEDICAL RESEARCH INST
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8152227
• 关键词: Multi; Level; Optimization; Membrane; Proteins

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 three classes of proteins: certain classes of transmembrane transporters, enzymes involved in lipid synthesis and lipid attachment to proteins, and complexes of seven-transmembrane segment proteins, including GPCRs, with single pass chaperone-like accessory proteins. The project also seeks to develop improved technologies for increasing levels of expression of functional TMPs in yeast, new approaches for specific fluorescent labeling of unpurified proteins, and the development of improved methods for biophysical characterization and screening of protein detergent complexes.

跨膜蛋白 (TMP) 占大多数基因组蛋白质编码潜力的 25% 以上。它们还在细胞和有机体生理学中发挥着核心作用，并且是大部分临床有用药物的靶标。然而，与可溶性蛋白质相比，我们对 TMP 的结构和功能的了解存在巨大缺陷。这主要归因于将 X 射线晶体学应用于 TMP 时通常遇到的重大障碍。该应用程序汇集了在分子生物学、生物化学、生物物理学和结构生物学（特别是膜蛋白）方面具有不同背景的三个独立的 PI，以创建用于 TMP 结构测定的管道。该管道的一个中心原则是需要在生产的最早阶段区分适合结构测定的蛋白质靶标和不适合结构测定的蛋白质靶标。考虑到这一点，我们建议针对直系同源、旁系同源和突变蛋白家族，在纯化和表征的早期阶段携带多种变体，以便最大限度地提高目标家族中最容易处理的成员成功结晶和衍射的机会。该项目利用细菌和酵母表达系统的现有克隆和表达方案，这些系统最适合并行表达策略，但将使用杆状病毒表达某些蛋白质。将使用小规模生长进行表达测试；将以中等规模生产给定目标的多种形式，以便使用现有的高通量筛选进行表征，以进行洗涤剂相容性、生物物理和生化表征以及小规模探索性结晶试验。只有来自中等规模分析的最有希望的候选者才会使用豪普特曼-伍德沃德研究所的设施进行大规模生产以进行高通量结晶筛选。将根据需要使用脂质立方相进行额外的筛选。除了 PSI 网络预期的蛋白质外，初始结构确定工作还将针对三类蛋白质：某些类别的跨膜转运蛋白、参与脂质合成和脂质附着到蛋白质的酶、以及七跨膜片段蛋白的复合物，包括 GPCR，与单通道伴侣蛋白样辅助蛋白。该项目还寻求开发改进技术，以提高酵母中功能性 TMP 的表达水平，对未纯化蛋白质进行特异性荧光标记的新方法，以及开发生物物理表征和蛋白质洗涤剂复合物筛选的改进方法。