Ultrafast photophysics of a positive reversibly switchable fluorescent protein

Fatima, Anam, He, Yongle, Iuliano, James Nicholas, Greetham, Gregory M., Malakar, Partha, Hall, Christopher R., Woroniecka, Helena A., Richardson, Brian C., French, Jarrod B., Lukacs, Andras, Tonge, Peter J. and Meech, Stephen R. (2025) Ultrafast photophysics of a positive reversibly switchable fluorescent protein. Chemical Science. ISSN 2041-6520

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Abstract

Reversibly switchable fluorescent proteins (rsFPs) are essential tools in super-resolution imaging. The mechanism operating in the widely applied negative switching rsFPs has been studied in detail. Much less attention has been paid to the positive switching rsFP variants, which offer the potential benefit of emissive states that do not photoswitch during measurement. Here we probe photochemical mechanism in all three photoactive states of the positive switching rsFP Kohinoor using a combination of ultrafast transient absorption, to probe chromophore population dynamics, and time resolved infrared, to access both chromophore populations and their effect on the surrounding protein matrix. We establish that none of the photochemical reactions are simple rate processes with transient absorption and transient IR data characterised by a common two component relaxation mechanism. Transient IR measurements reveal instantaneous coupling between the electronically excited chromophore and its protein environment, indicating that coupling arises from electrostatic or H-bonded interactions. In both on- and off- switching states the early phase of the excited state dynamics involve an initial relaxation in the perturbed protein environment, which leads to an intermediate state from which chromophore isomerization occurs. This result suggests that the protein dynamics play an active role in steering the excited state reaction, which is in-turn consistent with the known key role of the protein environment in tuning FP photophysics. Identifying and modifying the interactions between chromophore and protein will provide a means to optimise rsFP performance and thus a basis for development of improved labels for super-resolution bioimaging.

Item Type:	Article
Additional Information:	Funding information: SRM acknowledges financial support from EPSRC (EP/X011410/1, EP/J009148/1). We are grateful for financial support from the Hungarian National Research and Innovation Office in the frame of the 2024-1.2.3-HU-RIZONT-2024-00063 grant. A.L. acknowledges funding from the Hungarian National Research and Innovation Office (K-137557). PJT and JBF acknowledge funding from the National Science Foundation (NSF) (MCB-1817837 and MCB-2042704). Y.H. and J.N.I were supported by a National Institutes of Health Chemistry-Biology Interface Training Grant (T32GM092714). The structural biology beamlines at NSLS-II AMX is funded by National Institutes of Health, National Institute of General Medical Sciences (NIGMS) through a Center Core P30 Grant (P30GM133893).
Uncontrolled Keywords:	3*,good paper, novelty, good journal, methodology similar to earlier papers ,/dk/atira/pure/researchoutput/REFrank/3_
Faculty \ School:	Faculty of Science > School of Chemistry, Pharmacy and Pharmacology
UEA Research Groups:	Faculty of Science > Research Groups > Centre for Photonics and Quantum Science Faculty of Science > Research Groups > Chemistry of Light and Energy
Depositing User:	LivePure Connector
Date Deposited:	07 Aug 2025 11:30
Last Modified:	16 Aug 2025 00:40
URI:	https://ueaeprints.uea.ac.uk/id/eprint/100103
DOI:	10.1039/D5SC04491J

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