title: Mechanistic insights into reversible photoactivation in proteins of the GFP family.
abstract: Light-controlled modification of the fluorescence emission properties of proteins of the GFP family is of crucial importance for many imaging applications including superresolution microscopy. Here, we have studied the reversibly photoswitchable fluorescent protein mIrisGFP using optical spectroscopy. By analyzing the pH dependence of isomerization and protonation equilibria and the isomerization kinetics, we have obtained insight into the coupling of the chromophore to the surrounding protein moiety and a better understanding of the photoswitching mechanism. A different acid-base environment of the chromophore's protonating group in its two isomeric forms, which can be inferred from the x-ray structures of IrisFP, is key to the photoswitching function and ensures that isomerization and protonation are correlated. Amino acids near the chromophore, especially Glu212, rearrange upon isomerization, and Glu212 protonation modulates the chromophore pK(a). In mIrisGFP, the cis chromophore protonates in two steps, with pK(cis) of 5.3 and 6, which is much lower than pK(trans) (>10). Based on these results, we have put forward a mechanistic scheme that explains how the combination of isomeric and acid-base properties of the chromophore in its protein environment can produce negative and positive photoswitching modes.
authors.affiliation: Institute of Applied Physics and Center for Functional Nanostructures, Karlsruhe Institute of Technology, Karlsruhe, Germany.
authors.forename: Susan
authors.initials: S
authors.lastname: Gayda
authors.forename: Karin
authors.initials: K
authors.lastname: Nienhaus
authors.forename: G Ulrich
authors.initials: GU
authors.lastname: Nienhaus
mesh_terms: D003116:Color; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008027:Light; D008958:Models, Molecular; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary / Q000528:radiation effects; D013057:Spectrum Analysis; D013237:Stereoisomerism; D013696:Temperature
chemical_list: D049452:Green Fluorescent Proteins