In vivo Photoregulierung von Proteinstruktur und -funktion durch genetisch codierte photoschaltbare Click Aminosäure
Zusammenfassung der Projektergebnisse
Optogenetics, which uses light to control membrane channels in genetically defined neurons, has transformed the field of neuroscience. Bringing optical control to proteins in general would be a greater challenge to impact cell and organismal biology. Methods to install light responsiveness into proteins in live cells is key to this goal. Although optical control via photocaging critical residues has been demonstrated and photo-caged unnatural amino acids (Uaas) can be genetically incorporated, such control is irreversible and induces localized perturbations around a single residue only. Instead, bioactivity often requires the modulation of protein domains. Protein conformation can be switched by the photoisomerizable azobenzene unit, which can be installed onto proteins in vitro through chemical modifications. However, non-specific reactions of this reactive unit inside cells prevent its usage in vivo. The strategy presented here is to install photoresponsiveness to a general protein by genetically incorporating a photoswitchable click amino acid (PSCaa) in live cells. The PSCaa reacts with an appropriately positioned Cys to selectively form a side chain bridge, which allows regulating protein conformation and function by light-induced isomerization. Previously this concept and feasibility has been demonstrated by introducing the PSCaa into peptides via chemical synthesis. To realize this method onto proteins in vivo, we need to be able to cotranslationally incorporate the PSCaa in live cells. Therefore, we expanded the the genetic code for the site-specific incorporation of the PSCaa into proteins in both E. coli and mammalian cells. Evolving a synthetase specifically for PSCaa turned out to be a great challenge, presumably due to its unique extra long and rigid side chain that is not present in any other amino acids. From the designed library, I successfully evolved multiple synthetase mutants and demonstrated the site-specific incorporation of the PSCaa into proteins in E. coli with high efficiency and fidelity. More importantly, the evolved synthetases were also successfully imported into mammalian cells to produce recombinant proteins with the PSCaa selectively incorporated. Moreover, I showed that the benzylchloride PSCaa spontaneously generated a covalent bridge with a nearby cysteine residue and that the resultant azobenzene bridge isomerized in response to light. This bridge formation is unique in that it does not require extra triggers such as light needed for the thiol-ene click reaction. The co-translational formation of such a bridge should greatly facilitate its application in cells and in vivo. Therefore, this new family of photoswitchable Uaas will prove valuable in optical control of protein conformation and bioactivity in live cells, thus providing a novel avenue for “optobiology”, the in vivo investigation of biological processes with light.
Projektbezogene Publikationen (Auswahl)
- Genetically Encoding Photoswitchable Click Amino Acids in Escherichia coli and Mammalian Cells. Angew. Chem., 126, 4013-4017, 2014; Angew. Chem. Int. Ed., 53, 3932-3936, 2014
Hoppmann, C.; Lacey, V. K.; Louie, G. V.; Wei, J.; Noel, J. P.; Wang, L.
(Siehe online unter https://doi.org/10.1002/ange.201400001)
