title: Real-time monitoring of chromophore isomerization and deprotonation during the photoactivation of the fluorescent protein Dronpa.
abstract: Dronpa is a photochromic green fluorescent protein (GFP) homologue used as a probe in super-resolution microscopy. It is known that the photochromic reaction involves cis/trans isomerization of the chromophore and protonation/deprotonation of its phenol group, but the sequence in time of the two steps and their characteristic time scales are still the subject of much debate. We report here a comprehensive UV-visible transient absorption spectroscopy study of the photoactivation mechanism of Dronpa, covering all relevant time scales from ∼100 fs to milliseconds. The Dronpa-2 variant was also studied and showed the same behavior. By carefully controlling the excitation energy to avoid multiphoton processes, we could measure both the spectrum and the anisotropy of the first photoactivation intermediate. We show that the observed few nanometer blue-shift of this intermediate is characteristic for a neutral cis chromophore, and that its anisotropy of ∼0.2 is in good agreement with the reorientation of the transition dipole moment expected upon isomerization. These data constitute the first clear evidence that trans → cis isomerization of the chromophore precedes its deprotonation and occurs on the picosecond time scale, concomitantly to the excited-state decay. We found the deprotonation step to follow in ∼10 μs and lead directly from the neutral cis intermediate to the final state.
authors.affiliation: Ecole Normale Supérieure-PSL Research University , Département de Chimie, 24 rue Lhomond, 75005 Paris, France.
authors.forename: Dheerendra
authors.initials: D
authors.lastname: Yadav
authors.forename: Fabien
authors.initials: F
authors.lastname: Lacombat
authors.forename: Nadia
authors.initials: N
authors.lastname: Dozova
authors.forename: Fabrice
authors.initials: F
authors.lastname: Rappaport
authors.forename: Pascal
authors.initials: P
authors.lastname: Plaza
authors.forename: Agathe
authors.initials: A
authors.lastname: Espagne
mesh_terms: D049452:Green Fluorescent Proteins / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D011522:Protons; D013237:Stereoisomerism; D014466:Ultraviolet Rays
chemical_list: D011522:Protons; D049452:Green Fluorescent Proteins