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

Sep 29, 2026

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Photoconvertible fluorescent proteins enable high-resolution bioimaging but photoswitching “blinking” can complicate analysis, especially on ultrafast timescales. This study investigates ancestrally derived Kaede-like proteins with varying photoconversion and photoswitching efficiencies using ultrafast electronic and vibrational spectroscopies. Near-UV excitation initiates off/on photoswitching and green-to-red photoconversion, with fastest negative photoswitching in acidic buffers. Off states rapidly return to bright on states via proton transfer, while FSRS tracks native cis relaxation in a less photoswitchable mutant; design strategies to suppress off/on switching and enhance photoconversion are proposed.

LanguageEnglish

CategoryResearch & Report

FormatPDF

Pages14

Reading Time35 min

AccessFree to download

Introduction

Photoconvertible fluorescent proteins and green-to-red pcFPs

Chromophore states, photoswitching “off”/“on”, and photoconversion complexity

Mutation-dependent optical and dynamic behaviors

Chemical Science [rsc.li/chemical-science](rsc.li/chemical-science) ISSN 2041-6539 EDGE ARTICLE Chong Fang et al. Ultrafast spectroscopic signatures for off-to-on photoswitchable species in a green-to-red photoconvertible ﬂuorescent protein Chemical Science EDGE ARTICLE Cite this: Chem. Sci., 2026, 17, 151 All publication charges for this article have been paid for by the Royal Society of Chemistry Received 18th August 2025 Accepted 29th November 2025 DOI: 10.1039/d5sc06279a[rsc.li/chemical-science](rsc.li/chemical-science) Ultrafast spectroscopic signatures for oﬀ-to-on photoswitchable species in a green-to-red photoconvertible ﬂuorescent protein Taylor D. Krueger, Cheng Chen and Chong Fang * 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. Introduction Photoconvertible 􀀁uorescent proteins (pcFPs) are instrumental bioprobes for advanced imaging techniques.1,2 The Kaede-like green-to-red pcFPs bearing the three-residue His-Tyr-Gly chromophore are particularly useful for bioimaging techniques like photoactivated localization microscopy (PALM) .3 Certain aspects of the green-to-red photoconversion mechanism are well understood, including the ubiquitous peptide cleavage occurring alongside the chromophore conjugation extension that eﬀectively redshi􀀁s the biomolecular optical spectra.4 However, due to the low photoconversion quantum yield and broad timescales proposed ranging from femtoseconds (fs) to milliseconds, pertinent key details regarding the photoconversion mechanism remain elusive. While there have been important modi􀀃cations to Kaede-like pcFPs for improving performance, unknown mechanistic aspects hinder the rational Department of Chemistry, Oregon State University, 153 Gilbert Hall, Corvallis, Oregon 97331, USA. E-mail: [Chong.Fang@oregonstate.edu](Chong.Fang@oregonstate.edu); Web: [https://fanglab](https://fanglab). [oregonstate.edu/](oregonstate.edu/) design of more advanced bioprobes with targeted optical properties. Many of these fascinating proteins are based on the same motif and

FAQs

What is the main goal of this study on green-to-red photoconvertible fluorescent proteins?

To identify the primary ultrafast events that govern off-to-on photoswitching and green-to-red photoconversion after near-UV excitation.

How do the experiments explain the fastest negative photoswitching behavior?

They show that cis/trans isomerization underlying negative photoswitching occurs fastest in acidic buffers under green-light irradiation, producing a distorted neutral off state tied to the initial green cis anionic population.

What mechanism is proposed for off-to-on photoswitching and how is it supported experimentally?

Off/on photoswitching likely involves excited-state proton transfer from the distorted chromophore, which competes with photoconversion; femtosecond transient absorption and FSRS across mutants and photoinduced states support this interpretation.

Sep 29, 2026

Upload by Patrick