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Recovering true FRET efficiencies from smFRET investigations requires triplet state mitigation.
Pati, Avik K; Kilic, Zeliha; Martin, Maxwell I; Terry, Daniel S; Borgia, Alessandro; Bar, Sukanta; Jockusch, Steffen; Kiselev, Roman; Altman, Roger B; Blanchard, Scott C.
Afiliación
  • Pati AK; Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN, USA.
  • Kilic Z; Department of Chemistry, Birla Institute of Technology and Science, Pilani, Rajasthan, India.
  • Martin MI; Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN, USA.
  • Terry DS; Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN, USA.
  • Borgia A; Department of Chemical Biology & Therapeutics, St. Jude Children's Research Hospital, Memphis, TN, USA.
  • Bar S; Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN, USA.
  • Jockusch S; Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN, USA.
  • Kiselev R; Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN, USA.
  • Altman RB; Department of Chemical Biology & Therapeutics, St. Jude Children's Research Hospital, Memphis, TN, USA.
  • Blanchard SC; Center for Photochemical Sciences and Department of Chemistry, Bowling Green State University, Bowling Green, OH, USA.
Nat Methods ; 21(7): 1222-1230, 2024 Jul.
Article en En | MEDLINE | ID: mdl-38877317
ABSTRACT
Single-molecule fluorescence resonance energy transfer (smFRET) methods employed to quantify time-dependent compositional and conformational changes within biomolecules require elevated illumination intensities to recover robust photon emission streams from individual fluorophores. Here we show that outside the weak-excitation limit, and in regimes where fluorophores must undergo many rapid cycles of excitation and relaxation, non-fluorescing, excitation-induced triplet states with lifetimes orders of magnitude longer lived than photon-emitting singlet states degrade photon emission streams from both donor and acceptor fluorophores resulting in illumination-intensity-dependent changes in FRET efficiency. These changes are not commonly taken into consideration; therefore, robust strategies to suppress excited state accumulations are required to recover accurate and precise FRET efficiency, and thus distance, estimates. We propose both robust triplet state suppression and data correction strategies that enable the recovery of FRET efficiencies more closely approximating true values, thereby extending the spatial and temporal resolution of smFRET.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Transferencia Resonante de Energía de Fluorescencia Idioma: En Revista: Nat Methods Asunto de la revista: TECNICAS E PROCEDIMENTOS DE LABORATORIO Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Transferencia Resonante de Energía de Fluorescencia Idioma: En Revista: Nat Methods Asunto de la revista: TECNICAS E PROCEDIMENTOS DE LABORATORIO Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos