Multi-Level Optimization of Membrane Proteins for Crystallography

跨膜蛋白（TMPs）占大多数基因组蛋白质编码潜力的25%以上。它们还在细胞和生物体生理学中发挥核心作用，并且是所有临床有用药物的大部分靶点。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的不足。这可以主要归因于在将X射线晶体学应用于TMP时通常遇到的实质性障碍。该应用程序汇集了三个独立的PI，具有不同的背景，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，以创建一个管道TMP结构测定。管道的一个中心原则是需要在生产的最早可能阶段区分适合结构测定的蛋白质靶点和不适合结构测定的蛋白质靶点。考虑到这一点，我们建议靶向直系同源、旁系同源和突变蛋白质家族，通过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靶家族中最易处理的成员成功结晶和衍射的机会。该项目利用现有的克隆和表达协议的细菌和酵母表达系统，最适合平行表达策略，但将使用杆状病毒表达的一些蛋白质。将使用小规模生长进行表达检测;将以中等规模生产多种形式的给定靶标，以允许使用现有的高通量筛选进行表征，用于去污剂相容性、生物物理和生物化学表征以及小规模探索性结晶试验。只有来自中间规模分析的最有希望的候选物才能进行大规模生产，使用Hauptman-Woodward研究所的设施进行高通量结晶筛选。根据需要，将使用无水立方相进行额外筛选。除了PSI网络预期的蛋白质外，最初的结构确定工作将针对三类蛋白质：某些类型的跨膜转运蛋白，参与脂质合成和脂质与蛋白质结合的酶，以及七跨膜片段蛋白质的复合物，包括GPCR，与单程伴侣样辅助蛋白。该项目还寻求开发提高酵母中功能性TMP表达水平的改进技术，未纯化蛋白质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。

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.