Mechanically switching single-molecule fluorescence of GFP by unfolding and refolding

Proceedings of the National Academy of Sciences of the United States of America
Ziad Ganim, Matthias Rief

Abstract

Green fluorescent protein (GFP) variants are widely used as genetically encoded fluorescent fusion tags, and there is an increasing interest in engineering their structure to develop in vivo optical sensors, such as for optogenetics and force transduction. Ensemble experiments have shown that the fluorescence of GFP is quenched upon denaturation. Here we study the dependence of fluorescence on protein structure by driving single molecules of GFP into different conformational states with optical tweezers and simultaneously probing the chromophore with fluorescence. Our results show that fluorescence is lost during the earliest events in unfolding, 3.5 ms before secondary structure is disrupted. No fluorescence is observed from the unfolding intermediates or the ensemble of compact and extended states populated during refolding. We further demonstrate that GFP can be mechanically switched between emissive and dark states. These data definitively establish that complete structural integrity is necessary to observe single-molecule fluorescence of GFP.

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Citations

Apr 17, 2020·Chemical Communications : Chem Comm·Yongliang WangHongbin Li
Jan 16, 2019·Biomolecules·Dhawal ChoudharyCiro Cecconi
Aug 24, 2019·Protein and Peptide Letters·Rouhollah VahabpourAzam Bolhassani
Nov 17, 2020·Macromolecular Rapid Communications·Hanna TraegerStephen Schrettl
Mar 17, 2021·Proceedings of the National Academy of Sciences of the United States of America·Marc-André LeBlancMarcelo C Sousa
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Mar 13, 2021·Annual Review of Biophysics·Lisa S FischerCarsten Grashoff
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Sep 2, 2021·Biophysics Reviews·Jorge Alegre-Cebollada
Oct 21, 2020·Biophysical Journal·Punam SonarCiro Cecconi
Nov 23, 2021·Biochemistry·Justin NwaforTanya L Schneider

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