Biosynthetic production of fully carbon-13 labeled retinal in E. coli for structural and functional studies of rhodopsins
The isomerization of a covalently bound retinal is an integral part of both microbial and animal rhodopsin function. As such, detailed structure and conformational changes in the retinal binding pocket are of significant interest and are studied in various NMR, FTIR, and Raman spectroscopy experiments, which commonly require isotopic labeling of retinal. Unfortunately, the de novo organic synthesis of an isotopically-labeled retinal is complex and often cost-prohibitive, especially for large scale expression required for solid-state NMR. We present the novel protocol for biosynthetic production of an isotopically labeled retinal ligand concurrently with an apoprotein in E. coli as a cost-effective alternative to the de novo organic synthesis. Previously, the biosynthesis of a retinal precursor, β-carotene, has been introduced into many different organisms. We extended this system to the prototrophic E. coli expression strain BL21 in conjunction with the inducible expression of a β-dioxygenase and proteo-opsin. To demonstrate the applicability of this system, we were able to assign several new carbon resonances for proteorhodopsin-bound retinal by using fully 13C-labeled glucose as the sole carbon source. Furthermore, we demonstrated that this biosynthetically produced retinal can be extracted from E. coli cells by applying a hydrophobic solvent layer to the growth medium and reconstituted into an externally produced opsin of any desired labeling pattern.
KeywordsBiosynthetic isotope labeling Rhodopsins Retinal chromophore Carbon-13 labeling NMR spectroscopy Vibrational spectroscopy
This research was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant No. RGPIN-2014-04547 to V.L. and Grant No. RGPIN-2018-04397 to L.S.B, and by Basic Science Research Program through the National Research Foundation of Korea (NRF) to K.-H.J., funded by the Ministry of Education (Grant No. 2018R1A6A1A03024940). Additionally, R.A.M. and M.E.W. were recipients of NSERC doctoral scholarship, and J.d.V. was a recipient of Ontario Graduate Scholarship (OGS). We want to thank Alicia Cronin for her help with the initial expression system testing.
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