[{"data":1,"prerenderedAt":-1},["ShallowReactive",2],{"detail-sidebar-cat-0-en-105":3,"doc-seo-445503-105":59,"doc-detail-445503-en":130},{"code":4,"msg":5,"data":6},0,"success",[7,13,18,23,28,33,38,43,48,51,55],{"id":8,"doc_module":4,"doc_module_name":9,"category_name":10,"show_sort_weight":11,"slug":12},1,"Document","Story & Novel",90,"story-novel",{"id":14,"doc_module":4,"doc_module_name":9,"category_name":15,"show_sort_weight":16,"slug":17},2,"Literature",80,"literature",{"id":19,"doc_module":4,"doc_module_name":9,"category_name":20,"show_sort_weight":21,"slug":22},4,"Exam",70,"exam",{"id":24,"doc_module":4,"doc_module_name":9,"category_name":25,"show_sort_weight":26,"slug":27},5,"Comic",60,"comic",{"id":29,"doc_module":4,"doc_module_name":9,"category_name":30,"show_sort_weight":31,"slug":32},6,"Technology",50,"technology",{"id":34,"doc_module":4,"doc_module_name":9,"category_name":35,"show_sort_weight":36,"slug":37},7,"Healthcare",40,"healthcare",{"id":39,"doc_module":4,"doc_module_name":9,"category_name":40,"show_sort_weight":41,"slug":42},8,"Research & Report",30,"research-report",{"id":44,"doc_module":4,"doc_module_name":9,"category_name":45,"show_sort_weight":46,"slug":47},9,"Religion & Spirituality",20,"religion-spirituality",{"id":46,"doc_module":4,"doc_module_name":9,"category_name":49,"show_sort_weight":46,"slug":50},"World Cup","world-cup",{"id":52,"doc_module":4,"doc_module_name":9,"category_name":53,"show_sort_weight":52,"slug":54},10,"Lifestyle","lifestyle",{"id":56,"doc_module":4,"doc_module_name":9,"category_name":57,"show_sort_weight":24,"slug":58},19,"General","general",{"code":4,"msg":60,"data":61},"ok",{"site_id":62,"language":63,"slug":64,"title":65,"keywords":66,"description":67,"schema_data":68,"social_meta":123,"head_meta":125,"extra_data":127,"updated_unix":129},105,"en","ultrafast-spectroscopic-signatures-for-off-to-on-photoswitchable-species-in-a-green-to-red-photoconvertible-fluorescent-protein","Ultrafast spectroscopic signatures for off-to-on photoswitchable species in a green-to-red photoconvertible fluorescent protein","","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.",{"@graph":69,"@context":122},[70,84,105],{"@type":71,"itemListElement":72},"BreadcrumbList",[73,77,79,82],{"item":74,"name":75,"@type":76,"position":8},"https://docshare.wps.com","Home","ListItem",{"item":78,"name":9,"@type":76,"position":14},"https://docshare.wps.com/document/",{"item":80,"name":40,"@type":76,"position":81},"https://docshare.wps.com/document/research-report/",3,{"item":83,"name":65,"@type":76,"position":19},"https://docshare.wps.com/document/ultrafast-spectroscopic-signatures-for-off-to-on-photoswitchable-species-in-a-green-to-red-photoconvertible-fluorescent-protein/445503/",{"url":83,"name":65,"@type":85,"image":86,"author":91,"headline":65,"publisher":94,"fileFormat":97,"inLanguage":63,"description":67,"dateModified":98,"datePublished":99,"encodingFormat":97,"isAccessibleForFree":100,"interactionStatistic":101},"DigitalDocument",{"url":87,"@type":88,"width":89,"height":90},"https://docshare.wps.com/thumbnails/ultrafast-spectroscopic-signatures-for-off-to-on-photoswitchable-species-in-a-green-to-red-photoconvertible-fluorescent-protein/445503.png","ImageObject",300,407,{"name":92,"@type":93},"Patrick","Person",{"url":74,"name":95,"@type":96},"DocShare","Organization","application/pdf","2026-10-04","2026-09-29",true,{"@type":102,"interactionType":103,"userInteractionCount":14},"InteractionCounter",{"@type":104},"ViewAction",{"@type":106,"mainEntity":107},"FAQPage",[108,114,118],{"name":109,"@type":110,"acceptedAnswer":111},"What is the main goal of this study on green-to-red photoconvertible fluorescent proteins?","Question",{"text":112,"@type":113},"To identify the primary ultrafast events that govern off-to-on photoswitching and green-to-red photoconversion after near-UV excitation.","Answer",{"name":115,"@type":110,"acceptedAnswer":116},"How do the experiments explain the fastest negative photoswitching behavior?",{"text":117,"@type":113},"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.",{"name":119,"@type":110,"acceptedAnswer":120},"What mechanism is proposed for off-to-on photoswitching and how is it supported experimentally?",{"text":121,"@type":113},"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.","https://schema.org",{"og:url":83,"og:type":124,"og:title":65,"og:site_name":95,"og:description":67},"article",{"robots":126,"canonical":83},"index,follow",{"doc_id":128,"site_id":62},445503,1791098400,{"code":4,"msg":5,"data":131},{"doc_id":128,"user_id":132,"nickname":92,"user_avatar":133,"doc_module":4,"category_id":39,"category_name":40,"doc_title":65,"doc_description":67,"doc_content":134,"file_id":135,"file_url":136,"file_type":137,"file_size":138,"view_count":14,"is_deleted":4,"is_public":8,"is_downloadable":8,"audit_status":8,"page_count":139,"language":140,"language_code":63,"site_id":62,"html_lang":63,"table_of_contents":141,"faqs":142,"seo_title":143,"seo_description":67,"update_tm":144,"read_time":145},549758146520,"https://ap-avatar.wpscdn.com/avatar/80002397d8c0411e94?_k=1775819394049821470","Chemical Science  \n[rsc.li/chemical-science](rsc.li/chemical-science)  \nISSN 2041-6539  \nEDGE ARTICLE  \nChong Fang et al.  \nUltrafast spectroscopic signatures for off-to-on photoswitchable species in a green-to-red photoconvertible fluorescent protein  \nChemical Science  \nEDGE ARTICLE  \nCite this: Chem. Sci., 2026, 17, 151  \nAll publication charges for this article have been paid for by the Royal Society of Chemistry  \nReceived 18th August 2025  \nAccepted 29th November 2025 DOI: 10.1039/d5sc06279a[rsc.li/chemical-science](rsc.li/chemical-science)  \nUltrafast spectroscopic signatures for off-to-on photoswitchable species in a green-to-red photoconvertible fluorescent protein  \nTaylor D. Krueger,  Cheng Chen  and Chong Fang  *  \nPhotoconvertible 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.  \nIntroduction  \nPhotoconvertible 􀀁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  \nDepartment 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/)  \ndesign of more advanced bioprobes with targeted optical properties.  \nMany of these fascinating proteins are based on the same motif and","cbCaisQVwIyFBAR2","https://ap.wps.com/l/cbCaisQVwIyFBAR2","pdf",2104306,14,"English","# Introduction\n## Photoconvertible fluorescent proteins and green-to-red pcFPs\n## Chromophore states, photoswitching “off”/“on”, and photoconversion complexity\n## Mutation-dependent optical and dynamic behaviors","[{\"question\":\"What is the main goal of this study on green-to-red photoconvertible fluorescent proteins?\",\"answer\":\"To identify the primary ultrafast events that govern off-to-on photoswitching and green-to-red photoconversion after near-UV excitation.\"},{\"question\":\"How do the experiments explain the fastest negative photoswitching behavior?\",\"answer\":\"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.\"},{\"question\":\"What mechanism is proposed for off-to-on photoswitching and how is it supported experimentally?\",\"answer\":\"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.\"}]","Ultrafast spectroscopic signatures for off-to-on photoswitchable species in a green-to-red photoconvertible fluorescent protein | PDF",1790712287,35]