Research-Genetically-Encoded Micro-viscosity Reporter

The micro-viscosity and crowding can regulate protein functions inside cells. Unfortunately, conventional GFP cannot report on the local viscosity, because their chromophores are shielded within the protein β-barrel. Surprisingly, we discovered that the bright-to-dark photo-switching kinetics of photochromic fluorescent proteins, Dronpa, is slowed down by increasing medium viscosity. This is the first report that the environment can exert a hydrodynamic effect on the chromophore of an FP.


Fig. 1. Micro-viscosity dependent photoswitching kinetics of photochromic fluorescent proteins. (a) The energy diagram of Dronpa. The bright state, the dark state, and their transitions are depicted. (b) The on- to off- photoswitching kinetics of Dronpa-3 is extremely sensitive to the glycerol concentrations. (c) Protein structure dynamics during Dronpa photoswitching. The chromophore’s cis-trans isomerization is accompanied by conformational motion of the protein β-barrel. (d) The switching kinetics can be used to report the local viscosity experienced by a specific protein such as H2B.

We attributed this unusual effect to protein-flexibility mediated coupling between chromophore’s cis-trans isomerization and the breathing dynamics of protein β-barrel (Fig. 1). Consistent with this mechanism, the switching kinetics of Dronpa-3, a structurally more flexible mutant, is found to exhibit more pronounced viscosity dependence. This effect thus offers a unique genetically-encoded viscosity reporter, revealing protein-specific information about intracellular environments. In a biological application, a remarkable heterogeneity of the microviscosity experienced by H2B is observed, which might be related to the recently reported heterogeneous chromatin compaction.

Fig. 2. A hybrid genetic-chemical molecular rotor whose fluorescence lifetime can report on protein-specific micro-environments. (a) Photophysics of Cy3. After excitation, Cy3 can also isomerize from trans- to cis- configuration through a torsional motion, bringing it back to the ground state without emission. (b) Construct of a hybrid genetic-chemical molecular rotor probe, eDHFR-TMP-Cy3, which can be targeted to specific protein or organelle.


Later we generated this switching idea to go beyond fluorescent proteins and into organic fluorophores. We showed that the fluorescence lifetime of Cy3 dye is sensitive to local viscosity, as a viscous medium will hinder the torsional motion on the potential surface of the excited state and hence prolong the fluorescence lifetime (Fig. 2). Then by coupling to the TMP-tag technology, we developed a genetic-chemical molecular rotor probe (eDHFR-TMP-Cy3) which is useful for evaluating a wider variety of protein or organelle-specific cellular micro-environments.

Relevant Papers:
1. Y.-T. Kao, X. Zhu and W. Min. "Protein flexibility mediated coupling between photoswitching kinetics and the surrounding viscosity of a photochromic fluorescent protein", Proc. Natl. Acad. Sci. USA, 109, 3220 (2012).[PDF]
2. E. Gatzogiannis, Z. Chen, L. Wei, R. Wombacher, Y.-T. Kao, G. Yefremov, V. C. Cornish and W. Min. "Mapping protein-specific micro-environments in live cells by fluorescence lifetime imaging of a hybrid genetic-chemical molecular rotor tag", Chem. Commun., 48, 8694 (2012).[PDF]