title: Tryptophan-based chromophore in fluorescent proteins can be anionic.
abstract: Cyan fluorescent proteins (CFP) with tryptophan66-based chromophore are widely used for live cell imaging. In contrast to green and red fluorescent proteins, no charged states of the CFP chromophore have been described. Here, we studied synthetic CFP chromophore and found that its indole group can be deprotonated rather easily (pKa 12.4).We then reproduced this effect in the CFP mCerulean by placing basic amino acids in the chromophore microenvironment. As a result, green-emitting variant with an anionic chromophore and key substitution Val61Lys was obtained. This is the first evidence strongly suggesting that tryptophan-based chromophores in fluorescent proteins can exist in an anionic charged state. Switching between protonated and deprotonated Trp66 in fluorescent proteins represents a new unexplored way to control their spectral properties.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
authors.forename: Karen S
authors.initials: KS
authors.lastname: Sarkisyan
authors.forename: Ilia V
authors.initials: IV
authors.lastname: Yampolsky
authors.forename: Kyril M
authors.initials: KM
authors.lastname: Solntsev
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Lukyanov
authors.forename: Konstantin A
authors.initials: KA
authors.lastname: Lukyanov
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Mishin
mesh_terms: D000838:Anions; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D000838:Anions; C485182:Cyan Fluorescent Protein; D049452:Green Fluorescent Proteins; D014364:Tryptophan