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

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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.