Protein-flexibility mediated coupling between photoswitching kinetics and surrounding viscosity of a photochromic fluorescent protein.
Recent advances in fluorescent proteins (FPs) have generated a remarkable family of optical highlighters with special light responses. Among them, Dronpa exhibits a unique capability of reversible light-regulated on-off switching. However, the environmental dependence of this photochromism is largely unexplored. Herein we report that the photoswitching kinetics of the chromophore inside Dronpa is actually slowed down by increasing medium viscosity outside Dronpa. This finding is a special example of an FP where the environment can exert a hydrodynamic effect on the internal chromophore. We attribute this effect to protein-flexibility mediated coupling where the chromophore's cis-trans isomerization during photoswitching is accompanied by conformational motion of a part of the protein β-barrel whose dynamics should be hindered by medium friction. Consistent with this mechanism, the photoswitching kinetics of Dronpa-3, a structurally more flexible mutant, is found to exhibit a more pronounced viscosity dependence. Furthermore, we mapped out spatial distributions of microviscosity in live cells experienced by a histone protein using the photoswitching kinetics of Dronpa-3 fusion as a contrast mechanism. This unique reporter should provide protein-specific information about the crowded intracellular environments by offering a genetically encoded microviscosity probe, which did not exist with normal FPs before.
Department of Chemistry, Columbia University, New York, NY 10027, USA.
Ya-Ting
YT
Kao
Xinxin
X
Zhu
Wei
W
Min
D019943:Amino Acid Substitution; D000818:Animals; D037421:Anthozoa; D003460:Crystallization; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000528:radiation effects; D017930:Genes, Reporter; D057809:HEK293 Cells; D006657:Histones / Q000737:chemistry; D006801:Humans; D007536:Isomerism; D007700:Kinetics; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D010777:Photochemistry; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000187:drug effects / Q000528:radiation effects; D012996:Solutions; D012997:Solvents / Q000494:pharmacology; D014466:Ultraviolet Rays; D014783:Viscosity
D005456:Fluorescent Dyes; D006657:Histones; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D012996:Solutions; D012997:Solvents