Ultrafast spectroscopic signatures for <i>off</i> -to- <i>on</i> photoswitchable species in a green-to-red photoconvertible fluorescent protein

Photoconvertible fluorescent proteins (pcFPs) have greatly advanced life sciences and cellular imaging with sub-diffraction resolution. A subset of Kaede-like pcFPs can reversibly photoswitch and irreversibly photoconvert, which yield intriguing properties for sophisticated bioimaging, yet blinking may complicate image analysis. Many investigations on such pcFPs lack transient information that can dictate their optical properties, especially on ultrafast timescales. We study a family of ancestrally derived pcFPs with varying photoconversion and photoswitching efficiencies based on the least evolved ancestor (LEA). With ultrafast electronic and vibrational spectroscopies that complement steady-state measurements, we dissect the primary events upon near-UV excitation of the native and photoswitched neutral chromophores, which initiates both <i>off</i> → <i>on</i> photoswitching and green-to-red photoconversion. We demonstrate that <i>cis</i> → <i>trans</i> isomerization underlying negative photoswitching occurs the fastest in acidic buffers upon green light irradiation, which forms a distorted neutral <i>off</i> state proportional to the initial green <i>cis</i> anionic population. Femtosecond transient absorption measurements reveal that this dynamic <i>off</i> state rapidly photoswitches back to the bright on state upon near-UV excitation, in contrast to the native form. With various mutants and photoinduced states, we find that <i>off</i> → <i>on</i> photoswitching likely involves excited state proton transfer from the distorted chromophore, which competes with photoconversion. In contrast, femtosecond stimulated Raman spectroscopy (FSRS) of the much less photoswitchable LEA-A69T tracks the efficient nonradiative relaxation of the native <i>cis</i> neutral chromophore. We propose rational design strategies to inhibit <i>off</i> → <i>on</i> photoswitching while improving the photoconversion efficiency of both neutral states. This work is envisioned to inspire more dynamic investigations of diverse photochromic FPs on ultrafast timescales.