Infrared spectroscopy reveals multi-step multi-timescale photoactivation in the photoconvertible protein archetype dronpa

Abstract

Photochromic fluorescent proteins play key roles in super-resolution microscopy and optogenetics. The light-driven structural changes that modulate the fluorescence involve both trans-to-cis isomerization and proton transfer. The mechanism, timescale and relative contribution of chromophore and protein dynamics are currently not well understood. Here, the mechanism of off-to-on-state switching in dronpa is studied using femtosecond-to-millisecond time-resolved infrared spectroscopy and isotope labelling. Chromophore and protein dynamics are shown to occur on multiple timescales, from picoseconds to hundreds of microseconds. Following excitation of the trans chromophore, a ground-state primary product is formed within picoseconds. Surprisingly, the characteristic vibrational spectrum of the neutral cis isomer appears only after several tens of nanoseconds. Further fluctuations in protein structure around the neutral cis chromophore are required to form a new intermediate, which promotes the final proton-transfer reaction. These data illustrate the interplay between chromophore dynamics and the protein environment underlying fluorescent protein photochromism.

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Fig. 1: Structure and interactions for the dronpa chromophore in off and on states.
Fig. 2: TRIR data and analysis for dronpa2 over the picosecond to 100 ms timescale.
Fig. 3: DFT-calculated infrared transition wavenumbers and intensities for the three most prominent bands of the cis and trans forms of the neutral chromophore in its natural, 1-13C- and 3-13C-labelled forms.
Fig. 4: Steady-state infrared spectra for the dronpa chromophore in pure cis and cis + trans photostationary states, providing an experimental check of the calculated spectral shifts in hydrogen-bonding and non-hydrogen-bonding solvents.
Fig. 5: Isotope effect on dronpa2 EADS recovered from a global analysis with a common set of rate constants, showing that the 1,702 cm−1 transient is associated with the chromophore C=O stretch.
Fig. 6: Proposed mechanism for off to on switching in dronpa2.

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Acknowledgements

S.R.M. acknowledges EPSRC for financial support (EP/N033647/1 and EP/M001997/1). P.J.T. acknowledges NSF for financial support (CHE-1223819). A.M. acknowledges the Japan Ministry of Education, Culture, Sports, Science and Technology Grant-in-aid for Scientific research on Innovative Areas: Resonance Bio. The authors acknowledge STFC for access to the Central Laser Facility. Calculations were performed on the High Performance Computing Cluster at the University of East Anglia.

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S.P.L., A.A.G., C.R.H., A.L. and J.N.I. measured and collected the data. S.P.L. and C.R.H. analysed the data. A.A.G. and J.N.I. grew and purified the samples. G.A.J. performed the DFT calculations. G.M.G. and P.D. built, developed and managed the ULTRA and LifeTime apparatus used in the measurements. A.M. designed the dronpa2 protein. P.J.T. and S.R.M. designed the experiment and wrote the paper, with discussion and editorial input from all authors

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Correspondence to Peter J. Tonge or Stephen R. Meech.

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Supplementary Figures 1–9, Supplementary Tables 1–5, Supplementary Methods, Supplementary Data, Supplementary Analysis

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Laptenok, S.P., Gil, A.A., Hall, C.R. et al. Infrared spectroscopy reveals multi-step multi-timescale photoactivation in the photoconvertible protein archetype dronpa. Nature Chem 10, 845–852 (2018). https://doi.org/10.1038/s41557-018-0073-0

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