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

Applicant Dr. Frank Bernhard
Subject Area Structural Biology
Biochemistry
Term from 2018 to 2022
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 404640060
 
Final Report Year 2023

Final Report Abstract

Cell-free (CF) expression reactions are open systems that allow precise definition of conditions and compound concentrations during protein synthesis. Complete control of the amino acid pool and low reaction volumes make them particularly useful for selective and combinatorial amino acid labeling. For NMR studies, protein samples are usually synthesized with highly efficient lysates from E. coli cells. Although many cellular proteins are removed during lysate preparation, some metabolic activity remains in the CF lysates. We have standardized the S30 (= centrifugation at 30,000 x g) lysate production from the E. coli strain A19 and we obtain reliable and reproducible quality and composition. By proteome analysis, we have determined the composition of our S30 lysate and a number of enzymes were identified that are potentially responsible for the observed residual scrambling of the amino acids L-Asn, L-Asp, L-Gln, L-Ala and L-Glu. The objective of this study was to systematically delete the genes encoding for these enzymes in the strain A19. The constructs should result into “cleaner” S30 lysates with absent or at least mostly reduced amino acid scrambling activity, thus better supporting NMR approaches. A variety of cumulative chromosomal mutations in different combinations were constructed in A19 and resulting S30 lysates were analyzed for reduced scrambling activity upon CF synthesis of labeled proteins. Potential problems with negative effects on the viability and growth kinetics of the mutants as well as on the CF protein synthesis efficiency of the resulting S30 lysates were considered. Out of all analyzed constructs, we finally selected the construct “Stablelabel” containing deletions of the genes asnA, ansA/B, glnA, aspC and ilvE as giving the best combination of reduced NMR label scrambling in S30 lysates, while still maintaining high CF protein synthesis efficiency. Generally, significant 15N-label transfer from L-Asp and L-Asn to other amino acids as observed with A19 S30 lysates is now eliminated with corresponding “Stablelabel” lysates. Still notable weak transfer from L-Glu to L-Ala can be suppressed with the inhibitor aminooxyacetate. Label scrambling from L-Gln to other amino acids could not be addressed as too many lysate enzymes appear to be involved in this reaction. However, L-Gln scrambling can be suppressed with the inhibitor 5-diazo-4-oxo-L-norvaline. The deletion of ilvE in “Stablelabel” further enables specific side-chain methyl group labelling of L-Leu, L-Val or L-Ile residues by using corresponding precursors. We could exemplify this approach by side-chain labelling of L-Val and L-Leu residues of the proton pump proteorhodopsin. To facilitate future NMR approaches with membrane proteins in lipid environments, we further provide a systematic study for the efficient CF formation of membrane protein/nanoparticle complexes.

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