Ultrafast Dynamics of a Green Fluorescent Protein Chromophore Analogue: Competition between Excited-State Proton Transfer and Torsional Relaxation

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Ultrafast Dynamics of a Green Fluorescent Protein Chromophore Analogue: Competition between Excited-State Proton Transfer and Torsional Relaxation, J. Phys. Chem. B2016120 (36), pp 9716–9722

 

Green fluorescent protein (GFP) from the jellyfish Aequoria victoria  and its numerous variants are widely used as genetically encodable fl uorescent markers in biological imaging. The fluorescence of GFP is due to a p hydroxybenzylidene−imidazolinone (p -HBDI) chromophore. In GFP at physiological pH, p-HBDI exists in both neutral and anionic forms at the hydroxyl group. The emissive species is, however, the anion, independently of the form initially excited, owing to fast deprotonation of the neutral form in the excited state. In solution, free p-HBDI is nonfluorescent, with an excited-state lifetime of ∼ 1 ps.

Fluorescence quenching is ascribed to torsional motions at the ethylene bridge, which are no longer hindered by the protein. High fl uorescence quantum yields may indeed be recovered by locking the ethylene bridge in a ring, or encapsulating the whole molecule in an RNA aptamer. The exact torsion coordinate of p-HBDI is, however, still under debate. Voliani et al. reported effi cient cis/trans photoisomerization in water. On the basis of the weak viscosity dependence of the excited-state dynamics of p-HBDI, Meech and co-workers proposed a volume-conserving, “ hula-twist”  coordinate (concerted torsion of adjacent single and double bonds). In contrast, theoretical studies suggest that hula-twist is energetically disfavored and that p-HBDI and related molecules decay by torsion about either the single or the double bond, depending on protonation state, substituents, and environment. When going from GFP to solution, the photophysics of p-HBDI therefore switches from excited-state proton transfer (ESPT) to torsional relaxation. p-HBDI torsion is, however, observed in some fl uorescent proteins other than GFP, due to diff erent chromophore− protein interactions. ON− OFF photoswitching in photochromic proteins such as Dronpa is, for instance, based on chromophore cis/trans photoisomerization. The competition between ESPT and torsion is, hence, at the heart of the photophysics of GFPrelated fluorescent proteins and their chromophores.

 

 

The present work shows that the light-induced reactivity of OHIM is controlled by the protonation state of the diethylamino group. When it is deprotonated (forms 1−3 ), the excited-state dynamics is dominated by torsion. Conversely, when it is protonated, ultrafast proton transfer to the solvent is observed, followed by cis/trans isomerization of the double-bond and ground-state reprotonation. This strong eff ect is likely due to the fact that protonation drastically reduces the electron donor character of the diethylamino group by deactivating the nitrogen lone pair. OHIM is in fact a “ push− pull”  conjugated system, in which the diethylamino nitrogen lone pair may be transferred toward the imidazolinone group. Such a charge transfer is expected to weaken the central ethylene double-bond and facilitate cis/trans isomerization. The dynamics of lightinduced torsion has actually been reported to correlate to the electron donor− acceptor character of molecular structures in a series of derivatives of the PYP (photoactive yellow protein) chromophore, a molecule closely related to p-HBDI.  As far as OHIM is concerned, we propose that protonation of the diethylamino group indirectly favors proton transfer by slowing down torsion, which can no longer compete efficiently. In conclusion, the present study on OHIM allowed us to explore the competition between ESPT and torsional relaxation for the first time in a single GFP chromophore analogue, just by varying the pH. This competition is central to the photophysics of GFP-related fl uorescent proteins and their chromophores.

 

N'hésitez pas à consulter le communiqué de presse relatif à cet article : La dynamique d'un chromophore fluorescent à l'étude !

 

Résumé: 

J. Phys. Chem. B2016120 (36), pp 9716–9722

 

The competition between excited-state proton transfer (ESPT) and torsion plays a central role in the photophysics of fluorescent proteins of the green fluorescent protein (GFP) family and their chromophores. Here, it was investigated in a single GFP chromophore analogue bearing o-hydroxy and p-diethylamino substituents, OHIM. The light-induced dynamics of OHIM was studied by femtosecond transient absorption spectroscopy, at different pH. We found that the photophysics of OHIM is determined by the electron-donating character of the diethylamino group: torsional relaxation dominates when the diethylamino group is neutral, whereas ultrafast ESPT followed by cis/trans isomerization and ground-state reprotonation are observed when the diethylamino group is protonated and therefore inactive as an electron donor.

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Ultrafast Dynamics of a Green Fluorescent Protein Chromophore Analogue: Competition between Excited-State Proton Transfer and Torsional Relaxation

 

Chatterjee, T, Lacombat F, Yadav D, Mandal M, Plaza P, Espagne A, Mandal PK

 

J. Phys. Chem. B2016120 (36), pp 9716–9722

 

DOI: 10.1021/acs.jpcb.6b05795