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

该提案旨在通过消除标记背景结合新的氨基酸位点链特异性标记方案，设计高效的无细胞（CF）裂解物用于蛋白质标记。然后，这些技术将应用于复杂膜蛋白（如gpcr和ABC转运蛋白）的结构和功能NMR表征。CF表达平台提供了快速、可靠的多种膜蛋白表达途径。然而，用于核磁共振研究的高效氨基酸特异性或组合蛋白质标记方案仍然受到标签混乱和氘/质子反交换问题的影响。提供抑制剂、化学处理或裂解物氘化不能提供令人满意的结果，因为它们大大降低了蛋白质的生产效率。因此，我们打算通过累积裂解物工程来定制用于NMR应用的CF表达平台，用于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，已经确定了在蛋白质标记过程中观察到的背景问题的残留酶。基因敲除突变以及内源性标记策略将以有效和经济的方式从裂解物中消除这些酶。然后将用模型可溶性蛋白和膜蛋白的标记验证精制裂解物制备方案。此外，我们打算开发新的氨基酸13C/2H位点链特异性标记方案，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反应量将为常规位点链特异性蛋白质标记提供价格合理且具有高度竞争力的平台，而不会引起代谢混乱。作为第一个里程碑，我们已经可以在蛋白质中仅显示Val残基的高效甲基位点链标记，这到目前为止只能通过CF表达实现。我们将对开发的方案进行分析，以特定标记具有挑战性的靶标，如ABC转运体TapL的Tmd0结构域和插入膜环境的ß - 1肾上腺素能受体。这些目标的高质量样品的高效CF生产已经建立，核磁共振光谱的分辨率将系统地提高，以允许结构研究和配体结合相互作用。该项目将扩展CF技术的组合，特别是特定的标记选项，它将为NMR社区提供有价值的工具，用于分析动力学，相互作用，甚至是膜蛋白等复杂大蛋白的结构特征。

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.