{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T16:58:48Z","timestamp":1790787528827,"version":"4.1.0"},"reference-count":51,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,9,27]],"date-time":"2026-09-27T00:00:00Z","timestamp":1790467200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,27]],"date-time":"2026-09-27T00:00:00Z","timestamp":1790467200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["advanced.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Advanced Science"],"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>Genetically encoded fluorogen\u2010activating tags enable conditional fluorescence, yet the structural and excited\u2010state mechanisms underlying ligand activation remain unclear. Here, we combine X\u2010ray crystallography, computational modelling, and fluorescence lifetime imaging microscopy (FLIM) to define the structural and photophysical basis of fluorogen activation in the Fluorescence\u2010Activating and Absorption\u2010Shifting Tag (FAST). Structures of apo and ligand\u2010bound FAST reveal the interface for fluorogen binding. In the apo state, N\u2010terminal residues occlude the pocket, whereas ligand binding displaces these elements to generate a solvent\u2010accessible cavity stabilized by ordered water molecules. Distinct hydroxybenzylidene rhodanine derivatives differentially tune interfacial geometry and excited\u2010state ensembles, producing single or multiexponential fluorescence lifetimes in living cells that primarily reflect modulation of excited\u2010state relaxation. Leveraging this ligand dependence, we utilize FAST as a chemically gated F\u00f6rster resonance energy transfer (FRET) acceptor that induces reversible, concentration\u2010dependent donor lifetime shortening that does not require additional external controls. Using ligand\u2010controlled FLIM\u2010FRET, we resolve supramodular organization within Membrane\u2010Associated Guanylate Kinase (MAGUK) scaffolds in live cells, establishing FAST as a reversible photophysical module for mapping molecular architecture.<\/jats:p>","DOI":"10.1002\/advs.77693","type":"journal-article","created":{"date-parts":[[2026,9,27]],"date-time":"2026-09-27T18:00:31Z","timestamp":1790532031000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Reversible, Chemically Gated FRET via Ligand\u2010Activated Acceptors"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-2310-7913","authenticated-orcid":false,"given":"Nivedita","family":"Singh","sequence":"first","affiliation":[{"name":"Centre For Neuroscience Indian Institute of Science  Bangalore India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9813-3631","authenticated-orcid":false,"given":"Smruti Ranjan","family":"Nayak","sequence":"additional","affiliation":[{"name":"Molecular Biophysics Unit Indian Institute of Science  Bangalore India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1375-2223","authenticated-orcid":false,"given":"Sanjay Kumar","family":"Mohanty","sequence":"additional","affiliation":[{"name":"Centre For Neuroscience Indian Institute of Science  Bangalore India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-3439-2669","authenticated-orcid":false,"given":"Aishwarya","family":"Hanumantharaju","sequence":"additional","affiliation":[{"name":"Centre For Neuroscience Indian Institute of Science  Bangalore India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-1925-0533","authenticated-orcid":false,"given":"Amala","family":"Shaju","sequence":"additional","affiliation":[{"name":"Centre For Neuroscience Indian Institute of Science  Bangalore India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9435-5237","authenticated-orcid":false,"given":"Mini","family":"Jose","sequence":"additional","affiliation":[{"name":"Cell and Tissue Imaging Center\u2010Light Microscopy St Jude Children's Research Hospital  Memphis USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9519-5055","authenticated-orcid":false,"given":"Aravind","family":"Penmatsa","sequence":"additional","affiliation":[{"name":"Molecular Biophysics Unit Indian Institute of Science  Bangalore India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2103-1653","authenticated-orcid":false,"given":"Deepak","family":"Nair","sequence":"additional","affiliation":[{"name":"Centre For Neuroscience Indian Institute of Science  Bangalore India"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,9,27]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms23084396"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1529\/biophysj.106.092841"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1364\/OE.14.012217"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.0c00419"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bpj.2008.12.1459"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.bioconjchem.6b00169"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1021\/ja9099328"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1038\/s42003\u2010024\u201006501\u20101"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.3390\/bios15050274"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrn2540"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467\u2010022\u201031912\u20101"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2006.05.016"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrn.2016.18"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/S1097\u20102765(01)00411\u20107"},{"key":"e_1_2_9_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/S1097\u20102765(01)00416\u20106"},{"key":"e_1_2_9_17_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2017.11.003"},{"key":"e_1_2_9_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.str.2012.12.014"},{"key":"e_1_2_9_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2020.166793"},{"key":"e_1_2_9_20_1","doi-asserted-by":"publisher","DOI":"10.1117\/1.1755721"},{"key":"e_1_2_9_21_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bpj.2011.11.2438"},{"key":"e_1_2_9_22_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.accounts.2c00098"},{"key":"e_1_2_9_23_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1513094113"},{"key":"e_1_2_9_24_1","doi-asserted-by":"publisher","DOI":"10.1039\/C7SC01364G"},{"key":"e_1_2_9_25_1","doi-asserted-by":"publisher","DOI":"10.1002\/chem.201901151"},{"key":"e_1_2_9_26_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.biochem.8b00649"},{"key":"e_1_2_9_27_1","doi-asserted-by":"publisher","DOI":"10.1039\/C8NR07809B"},{"key":"e_1_2_9_28_1","doi-asserted-by":"publisher","DOI":"10.1038\/s42003\u2010022\u201003662\u20109"},{"key":"e_1_2_9_29_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms231911361"},{"key":"e_1_2_9_30_1","doi-asserted-by":"publisher","DOI":"10.1039\/D1SC01454D"},{"key":"e_1_2_9_31_1","doi-asserted-by":"publisher","DOI":"10.1002\/chem.202101250"},{"key":"e_1_2_9_32_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598\u2010017\u201012400\u20109"},{"key":"e_1_2_9_33_1","doi-asserted-by":"publisher","DOI":"10.1002\/chem.202004760"},{"key":"e_1_2_9_34_1","doi-asserted-by":"publisher","DOI":"10.1023\/A:1022995327718"},{"key":"e_1_2_9_35_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10895\u2010014\u20101368\u20101"},{"key":"e_1_2_9_36_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0509922103"},{"key":"e_1_2_9_37_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.apsb.2018.02.001"},{"key":"e_1_2_9_38_1","doi-asserted-by":"publisher","DOI":"10.2174\/1386207319666160408150320"},{"key":"e_1_2_9_39_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1612963113"},{"key":"e_1_2_9_40_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi201178v"},{"key":"e_1_2_9_41_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.pep.2009.07.007"},{"key":"e_1_2_9_42_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M117.782490"},{"key":"e_1_2_9_43_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.medpho.2014.11.001"},{"key":"e_1_2_9_44_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.rechem.2026.103733"},{"key":"e_1_2_9_45_1","doi-asserted-by":"publisher","DOI":"10.1007\/s11068\u2010008\u20109024\u20109"},{"key":"e_1_2_9_46_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.2021"},{"key":"e_1_2_9_47_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature12533"},{"key":"e_1_2_9_48_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.drudis.2010.10.003"},{"key":"e_1_2_9_49_1","unstructured":"S.Dallakyan \u201cMGLTools version 1.5.6 \u201d (2010)."},{"key":"e_1_2_9_50_1","doi-asserted-by":"publisher","DOI":"10.1186\/1758-2946-3-33"},{"key":"e_1_2_9_51_1","doi-asserted-by":"publisher","DOI":"10.1002\/jcc.21256"},{"key":"e_1_2_9_52_1","doi-asserted-by":"publisher","DOI":"10.1021\/ci200227u"}],"container-title":["Advanced Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/advanced.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/advs.77693","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/advanced.onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/advs.77693","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/advanced.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/advs.77693","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T16:03:14Z","timestamp":1790784194000},"score":1,"resource":{"primary":{"URL":"https:\/\/advanced.onlinelibrary.wiley.com\/doi\/10.1002\/advs.77693"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,27]]},"references-count":51,"alternative-id":["10.1002\/advs.77693"],"URL":"https:\/\/doi.org\/10.1002\/advs.77693","archive":["Portico"],"relation":{},"ISSN":["2198-3844","2198-3844"],"issn-type":[{"value":"2198-3844","type":"print"},{"value":"2198-3844","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,27]]},"assertion":[{"value":"2026-03-16","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-09-02","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-09-27","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e77693"}}