Customizing cell-free labelling strategies for the NMR analysis of membrane proteins

定制用于膜蛋白 NMR 分析的无细胞标记策略

• 批准号: 404640060
• 负责人: Dr. Frank Bernhard
• 金额: --
• 依托单位: Institut für Biophysikalische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/404640060?language=en
• 关键词: Customizing; cell; free; labelling; strategies

The proposal aims to design highly productive cell-free (CF) lysates for protein labelling by eliminating labelling backgrounds combined with new amino acid site-chain specific labelling schemes. The technologies will then be applied to the structural and functional NMR characterization of difficult membrane proteins such as GPCRs and ABC transporters. CF expression platforms provide fast and reliable access to a wide variety of membrane proteins. However, efficient amino acid specific or combinatorial protein labelling schemes for NMR studies are still compromised by label scrambling and deuterium/proton back exchange problems. Supplying inhibitors, chemical treatment or lysate deuteration do not provide satisfying results as they considerable reduce protein production efficiencies. We therefore intend to customize CF expression platforms to NMR applications by cumulated lysate engineering for amino acid stabilization and label background suppression. Based on our recent lysate proteome analysis, residual enzymes responsible for the observed background problems during protein labelling have been identified. Genetic knock-out mutations as well as endogenous tagging strategies will be combined to eliminate these enzymes in an efficient and cost-effective way from the lysates. Refined lysate preparation protocols will then be verified with the labelling of model soluble and membrane proteins.We furthermore intend to develop new protocols for the 13C/2H site-chain specific labelling of amino acids, primarily targeting methyl groups in Val, Leu and Ile residues. Residual metabolic pathway fragments in the lysates will be analyzed for their functionality, complemented if appropriate, and activated by supplied specific precursors. The low CF reaction volumes will then provide affordable and highly competitive platforms for routine site-chain specific protein labelling devoid of metabolic scrambling. As a first milestone, we can already show the efficient methyl site-chain labeling of only Val residues in proteins, which is so far only possible by CF expression.The developed protocols we be will analyzed for the specific labelling of challenging targets such as the Tmd0 domain of the ABC transporter TapL and the ß1-adrenergic receptor inserted into membrane environments. Efficient CF production of high quality samples of these targets have already been established and resolution of NMR spectra will be systematically improved to allow structural studies and ligand binding interactions. The project will generally expand the portfolio of CF technologies in particular for specific labelling options and it will provide valuable tools to the NMR community for analyzing dynamics, interactions and even structural features of difficult large proteins such as membrane proteins.

该提案旨在通过消除标记背景并结合新的氨基酸位点链特异性标记方案，设计用于蛋白质标记的高产无细胞（CF）裂解液。然后，这些技术将应用于 GPCR 和 ABC 转运蛋白等困难膜蛋白的结构和功能 NMR 表征。 CF 表达平台可快速、可靠地获取多种膜蛋白。然而，用于 NMR 研究的有效氨基酸特异性或组合蛋白质标记方案仍然受到标记扰乱和氘/质子反向交换问题的影响。提供抑制剂、化学处理或裂解物氘化并不能提供令人满意的结果，因为它们会大大降低蛋白质生产效率。因此，我们打算通过累积裂解物工程来定制 CF 表达平台，以适应 NMR 应用，以实现氨基酸稳定和标签背景抑制。根据我们最近的裂解物蛋白质组分析，已经确定了导致蛋白质标记过程中观察到的背景问题的残留酶。基因敲除突变和内源标记策略将结合起来，以有效且经济有效的方式从裂解物中消除这些酶。然后，将通过模型可溶性蛋白和膜蛋白的标记来验证精制裂解液制备方案。此外，我们还打算开发用于氨基酸 13C/2H 位点链特异性标记的新方案，主要针对 Val、Leu 和 Ile 残基中的甲基。将分析裂解物中残留的代谢途径片段的功能，在适当的情况下进行补充，并由提供的特定前体激活。低 CF 反应体积将为常规位点链特异性蛋白质标记提供经济实惠且极具竞争力的平台，而无需代谢扰乱。作为第一个里程碑，我们已经可以展示蛋白质中仅 Val 残基的有效甲基位点链标记，这迄今为止只能通过 CF 表达实现。我们开发的方案将分析具有挑战性的目标的特定标记，例如 ABC 转运蛋白 TapL 的 Tmd0 结构域和插入膜环境中的 ß1-肾上腺素能受体。这些靶标的高质量样品的高效 CF 生产已经建立，并且 NMR 谱的分辨率将得到系统提高，以进行结构研究和配体结合相互作用。该项目总体上将扩展 CF 技术组合，特别是针对特定标记选项，并将为 NMR 界提供有价值的工具，用于分析困难的大蛋白（如膜蛋白）的动力学、相互作用甚至结构特征。