Ultrafast spectroscopic signatures for off-to-on photoswitchable species in a green-to-red photoconvertible...

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 off → on photoswitching and green-to-red photoconversion. We demonstrate that cis → trans isomerization underlying negative photoswitching occurs the fastest in acidic buffers upon green light irradiation, which forms a distorted neutral off state proportional to the initial green cis anionic population. Femtosecond transient absorption measurements reveal that this dynamic off 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 off → on 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 cis neutral chromophore. We propose rational design strategies to inhibit off → on 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.