Computational redesign of a fluorogen activating protein with Rosetta
Fig 2
Experimental characterization of new proteins in cellulo.
(A-D) Representative widefield fluorescence images of living HEK293 cells transiently transfected with H2B-TagBFP-DiB1 (A, B) or H2B-TagBFP-DiB-RM (C, D) constructs in presence of 0.5 μM M739 in green (A, C) and blue (B, D) detection channels; scale bars are 15 μm. (E) Green to blue fluorescence signal ratio distribution in nuclei of living HEK293 cells transiently transfected with H2B-DiB1-TagBFP or H2B-DiB-RM-TagBFP in presence of 0.5 μM M739. Represented are data for 307 cells transiently transfected with H2B-DiB1-TagBFP and 241 cells transiently transfected with H2B-DiB-RM-TagBFP. (F-G) Confocal fluorescence microscopy imaging (excitation: 488 nm, emission: 520–560 nm) of living HeLa cells transiently transfected with DiB-RM constructs in presence of 0.25 μM M739; (F) vimentin-DiB-RM, scale bar 10 μm; (G) LifeAct-TagBFP-DiB-RM, scale bar 20 μm. (H-I) Widefield fluorescence images of living HEK293 cells transiently cotransfected with H2B-DiB-RM-splitN1-109 and DiB-RM-splitC110-177-TagBFP constructs in presence of 0.1 μM M739 in green (H) and blue (I) detection channels; scale bars are 15 μm.