title: Cis-trans photoisomerization of fluorescent-protein chromophores.
abstract: Photochromic variants of fluorescent proteins are opening the way to a number of opportunities for high-sensitivity regioselective studies in the cellular environment and may even lead to applications in information and communication technology. Yet, the detailed photophysical processes at the basis of photoswitching have not been fully clarified. In this paper, we used synthetic FP chromophores to clarify the photophysical processes associated with the photochromic behavior. In particular, we investigated the spectral modification of synthetic chromophore analogues of wild-type green fluorescent protein (GFP), Y66F GFP (BFPF), and Y66W GFP (CFP) upon irradiation in solutions of different polarities. We found that the cis-trans photoisomerization mechanism can be induced in all the chromophores. The structural assignments were carried out both by NMR measurements and DFT calculations. Remarkably, we determined for the first time the spectra of neutral trans isomers in different solvents. Finally, we calculated the photoconversion quantum yields by absorption measurements under continuous illumination at different times and by a nanosecond laser-flash photolysis method. Our results indicate that cis-trans photoisomerization is a general mechanism of FP chromophores whose efficiency is modulated by the detailed mutant-specific protein environment.
authors.affiliation: Scuola Normale Superiore, Italian Institute of Technology, Pisa, Italy. v.voliani@sns.it
authors.forename: Valerio
authors.initials: V
authors.lastname: Voliani
authors.forename: Ranieri
authors.initials: R
authors.lastname: Bizzarri
authors.forename: Riccardo
authors.initials: R
authors.lastname: Nifosì
authors.forename: Stefania
authors.initials: S
authors.lastname: Abbruzzetti
authors.forename: Elena
authors.initials: E
authors.lastname: Grandi
authors.forename: Cristiano
authors.initials: C
authors.lastname: Viappiani
authors.forename: Fabio
authors.initials: F
authors.lastname: Beltram
mesh_terms: D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D008164:Luminescent Proteins / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D015394:Molecular Structure; D010777:Photochemistry; D010782:Photolysis / Q000528:radiation effects; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism; D013816:Thermodynamics
chemical_list: C485182:Cyan Fluorescent Protein; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; C409504:blue fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins