In the latest issue of ChemBioChem, G. Charbon et al. reported a technique for in-vivo fluorescent labeling of proteins without improper protein assembly and/or function. They successfully placed very small fluorescent tag into the bacterial tubulin, FtsZ. The fluorescent, coumarin-derived amino acid (CouAA, see structure above), is encoded into a given sequence. CouAA is transfered to a growing polypeptide when nonsense amber stop codons UAG are present, and the expression of genes terminated by amber codons is not affected.
I think this research and Prof.Deiters' are somewhat alike. I was thinking about the unnatural AAs may affect the structure of proteins when Prof.Deiters gave the speech at the new graduate orientation.
ReplyDeleteThe original paper describing the incorporation of this coumarin amino acid is a little older. Issues that need to be addressed: 1) incorporate fluorophores with better photochemical properties, and 2) incorporate fluorophores in live mammalian cells. I know that several labs are working on this.
ReplyDeleteIncorporation of fluorescent AAs in live mammalian cells would be a nice shortcut towards protein research, since a strong reporting system would not need to be developed for every application. Producing fluorescent proteins with relatively small structural changes could be very advantageous towards maintaining biological relevance over systems that use tags, probes, linkers etc.
ReplyDeleteI wouldn't be extremely surprised if most organisms have at least one active enzyme that would glycosylate, methylate, sulfate, etc. at the 7-OH of this fluorophore, quenching the fluorescence. I'm certain you wouldn't have success labeling plant proteins with this; there are dozens of arabidopsis GTs that glycosylate the 7-OH of related coumarins, like esculetin.
ReplyDeleteA more general probe would probably need a more bio-orthogonal fluorophore.