CE11 - Caractérisation des structures et relations structure-fonctions des macromolécules biologiques

Ultrafast structural dynamics of Archaerhodopsin-3 in its fluorescent state – ULTRArchea

Submission summary

Archaerhodopsin-3 (AR-3), a photo-activated proton pump from Halorubrum sodomense, is the progenitor of an entire class of genetically encoded fluorescent membrane voltage indicators (GEVI). Like in other microbial rhodopsins, absorption of visible light triggers in AR-3 the retinal chromophore reversible photoisomerization which leads to the existence of several intermediates with lifetimes covering the sub-picosecond to millisecond time scale. AR-3-based applications in neuron microscopy are being actively diversified, thanks to extensive genetic modifications and screening of the best GEVIs. But their range of application is limited by their rather low fluorescence brightness, when compared to fluorescent proteins (FPs). Along with the genetic selection approach, the details of the molecular fluorescence mechanisms of archaerhodopsins were only poorly studied, which hinders a rational approach to improving the fluorescence properties. At variance with other 1-photon-based fluorescent rhodopsins and FPs, wild-type AR-3 fluorescence is taking place after 3-photon absorption. The fluorescent state is produced at a late stage of the AR-3 photo-cycle (> 1 ms) from an intermediate photo-product in the ground state after excited state picosecond isomerization, hence making its structural or spectroscopic characterization very difficult. Hence, a detailed understanding is lacking so far.
This challenge will be taken up within the UltrArchae research project by applying advanced methods of ultrafast electronic and vibrational spectroscopy and state-of-the-art quantum chemistry methods, combined with time-resolved X-ray crystallography. These will be applied to characterize the several transient intermediates characterizing most of the AR-3 photo-cycle. These methods, in particular a novel excitation scheme with three precisely timed ultrashort laser pulses and a new QM/MM model based on recently developed mixed-reference spin-flip time-dependent density functional theory, will allow obtaining detailed information on the structural dynamics and excited state energy profile of the key meta-stable and fluorescent conformational states (“intermediates”), for AR-3 and designed mutants. In particular, point mutations on specific positions (proton acceptors, retinal binding pocket) will help to understand what are the most affected AR-3 photocycle steps: improved primary photoisomerization increasing the concentration of the intermediates, modification of the lifetime of the fluorescent intermediate by playing on protein cage interactions with the chromophore or changing the chemical equilibrium between these intermediates, or activation/deactivation of specific retinal decay channels in the fluorescent intermediate state by mutation of residues.
UltrArchae is an interdisciplinary project that gathers physicists, physical chemists and molecular biologists from France and UK. It will provide for the first time a detailed mechanistic understanding of the structure-function relationship leading to unusual fluorescence properties in AR-3 and its variants. We anticipate that these findings will be useful and of general importance for the further improvement of fluorescent sensors based on retinal proteins.

Project coordination

Stefan HAACKE (Institut de physique et chimie des matériaux de Strasbourg (UMR 7504))

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


LASIRe Laboratoire Avancé de Spectroscopie pour les Intéractions la Réactivité et l'Environnement
ICR Institut de Chimie Radicalaire
National Physical Laboratory, UK / Department Chemical Biology / BioMetrology group
University of Oxford / Biochemistry Department
IPCMS Institut de physique et chimie des matériaux de Strasbourg (UMR 7504)

Help of the ANR 583,501 euros
Beginning and duration of the scientific project: December 2021 - 48 Months

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