Longteng Tang, Riley M Bednar, Nikita D Rozanov, Marcus L Hemshorn, Ryan A Mehl, Chong Fang
{"title":"四嗪编码绿色荧光蛋白高生物正交荧光性的合理设计。","authors":"Longteng Tang, Riley M Bednar, Nikita D Rozanov, Marcus L Hemshorn, Ryan A Mehl, Chong Fang","doi":"10.1002/ntls.20220028","DOIUrl":null,"url":null,"abstract":"<p><p>The development of bioorthogonal fluorogenic probes constitutes a vital force to advance life sciences. Tetrazine-encoded green fluorescent proteins (GFPs) show high bioorthogonal reaction rate and genetic encodability, but suffer from low fluorogenicity. Here, we unveil the real-time fluorescence mechanisms by investigating two site-specific tetrazine-modified superfolder GFPs via ultrafast spectroscopy and theoretical calculations. Förster resonance energy transfer (FRET) is quantitatively modeled and revealed to govern the fluorescence quenching; for GFP150-Tet with a fluorescence turn-on ratio of ~9, it contains trimodal subpopulations with good (P<sub>1</sub>), random (P<sub>2</sub>), and poor (P<sub>3</sub>) alignments between the transition dipole moments of protein chromophore (donor) and tetrazine tag (Tet-v2.0, acceptor). By rationally designing a more free/tight environment, we created new mutants Y200A/S202Y to introduce more P<sub>2</sub>/P<sub>1</sub> populations and improve the turn-on ratios to ~14/31, making the fluorogenicity of GFP150-Tet-S202Y the highest among all up-to-date tetrazine-encoded GFPs. In live eukaryotic cells, the GFP150-Tet-v3.0-S202Y mutant demonstrates notably increased fluorogenicity, substantiating our generalizable design strategy.</p>","PeriodicalId":74244,"journal":{"name":"Natural sciences (Weinheim, Germany)","volume":null,"pages":null},"PeriodicalIF":2.6000,"publicationDate":"2022-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9699285/pdf/nihms-1827592.pdf","citationCount":"4","resultStr":"{\"title\":\"Rational Design for High Bioorthogonal Fluorogenicity of Tetrazine-Encoded Green Fluorescent Proteins.\",\"authors\":\"Longteng Tang, Riley M Bednar, Nikita D Rozanov, Marcus L Hemshorn, Ryan A Mehl, Chong Fang\",\"doi\":\"10.1002/ntls.20220028\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>The development of bioorthogonal fluorogenic probes constitutes a vital force to advance life sciences. Tetrazine-encoded green fluorescent proteins (GFPs) show high bioorthogonal reaction rate and genetic encodability, but suffer from low fluorogenicity. Here, we unveil the real-time fluorescence mechanisms by investigating two site-specific tetrazine-modified superfolder GFPs via ultrafast spectroscopy and theoretical calculations. Förster resonance energy transfer (FRET) is quantitatively modeled and revealed to govern the fluorescence quenching; for GFP150-Tet with a fluorescence turn-on ratio of ~9, it contains trimodal subpopulations with good (P<sub>1</sub>), random (P<sub>2</sub>), and poor (P<sub>3</sub>) alignments between the transition dipole moments of protein chromophore (donor) and tetrazine tag (Tet-v2.0, acceptor). By rationally designing a more free/tight environment, we created new mutants Y200A/S202Y to introduce more P<sub>2</sub>/P<sub>1</sub> populations and improve the turn-on ratios to ~14/31, making the fluorogenicity of GFP150-Tet-S202Y the highest among all up-to-date tetrazine-encoded GFPs. In live eukaryotic cells, the GFP150-Tet-v3.0-S202Y mutant demonstrates notably increased fluorogenicity, substantiating our generalizable design strategy.</p>\",\"PeriodicalId\":74244,\"journal\":{\"name\":\"Natural sciences (Weinheim, Germany)\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.6000,\"publicationDate\":\"2022-10-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9699285/pdf/nihms-1827592.pdf\",\"citationCount\":\"4\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Natural sciences (Weinheim, Germany)\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1002/ntls.20220028\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2022/8/8 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q2\",\"JCRName\":\"MULTIDISCIPLINARY SCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Natural sciences (Weinheim, Germany)","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1002/ntls.20220028","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2022/8/8 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"MULTIDISCIPLINARY SCIENCES","Score":null,"Total":0}
Rational Design for High Bioorthogonal Fluorogenicity of Tetrazine-Encoded Green Fluorescent Proteins.
The development of bioorthogonal fluorogenic probes constitutes a vital force to advance life sciences. Tetrazine-encoded green fluorescent proteins (GFPs) show high bioorthogonal reaction rate and genetic encodability, but suffer from low fluorogenicity. Here, we unveil the real-time fluorescence mechanisms by investigating two site-specific tetrazine-modified superfolder GFPs via ultrafast spectroscopy and theoretical calculations. Förster resonance energy transfer (FRET) is quantitatively modeled and revealed to govern the fluorescence quenching; for GFP150-Tet with a fluorescence turn-on ratio of ~9, it contains trimodal subpopulations with good (P1), random (P2), and poor (P3) alignments between the transition dipole moments of protein chromophore (donor) and tetrazine tag (Tet-v2.0, acceptor). By rationally designing a more free/tight environment, we created new mutants Y200A/S202Y to introduce more P2/P1 populations and improve the turn-on ratios to ~14/31, making the fluorogenicity of GFP150-Tet-S202Y the highest among all up-to-date tetrazine-encoded GFPs. In live eukaryotic cells, the GFP150-Tet-v3.0-S202Y mutant demonstrates notably increased fluorogenicity, substantiating our generalizable design strategy.