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.
