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）占大多数基因组的蛋白质编码潜力的25％以上。它们还在细胞和生物生理学中起着核心作用，并且是所有临床有用药物中很大一部分的靶标。但是，与可溶性蛋白相比，我们对TMP的结构和功能的了解存在巨大缺陷。这主要归因于在将X射线晶体学应用于TMP时通常遇到的实质性障碍。该应用汇集了三个独立的PI，具有分子生物学，生物化学，生物物理学和结构生物学，特别是膜蛋白的不同背景，以创建用于TMP结构确定的管道。管道的中心宗旨是需要在蛋白质靶标之间最早的生产阶段进行区分，这些蛋白质靶标与结构确定和没有结构的确定。考虑到这一点，我们建议靶向直系同源，寄生虫和突变的蛋白质家族，并在纯化和表征的早期阶段携带多种变体，以最大程度地提高目标家族中最可触及的成员到成功结晶和衍射的机会。该项目利用了最适合平行表达策略的细菌和酵母表达系统的现有克隆和表达方案，但将使用Bacculovirus表达来进行某些蛋白质。表达测试将使用小规模生长进行；给定目标的多种形式将以中等规模产生，以允许使用现有的高通量屏幕来表征清洁剂的兼容性，生物物理和生化特征以及小规模的探索性结晶试验。只有中等规模分析中最有希望的候选人才能使用Hauptman-Woodward Institute的设施进行大规模生产，以进行高通量结晶筛查。将根据需要进行使用脂质立方相进行额外的筛查。除了从PSI网络中预期的蛋白质外，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，参与脂质合成涉及的酶以及对蛋白质的脂质附着的酶，以及七跨膜蛋白的络合物，包括具有单个Pass Choperone蛋白的GPCR，包括单个Pass Chaperone蛋白，类似于GPCR。该项目还旨在开发改进的技术，以提高酵母中功能性TMP的表达水平，未育蛋白的特定荧光标记的新方法，以及开发改进的生物物理表征和筛选蛋白质洗涤剂复合物的方法。