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.

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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.

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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]