Optical Control of Protein Activity by Fluorescent Protein Domains
Xin X. Zhou1,
Hokyung K. Chung1,
Amy J. Lam1, and
Michael Z. Lin1,2,*
1 Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
2 Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
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Fig. 1. Control of photochromic FP domain association by light. (A) Hypothesized bidirectional control of Dronpa145N oligomerization state by 500-nm cyan and 400-nm violet light. (B) Native PAGE of Dronpa145N (100 μM) demonstrated 500-nm–induced dissociation and 400-nm–induced retetramerization. mRuby2 (31), tdTomato, and dsRed2 (20 μM) served as monomeric, dimeric, and tetrameric standards, respectively. All proteins were polyhistidine-tagged at the NT. (C) Absorbance spectra confirm reversible photoswitching. (D) Hypothesized bidirectional conformational switching by light in a Dronpa145K-Dronpa145N (K-N) tandem dimer. (E) Native PAGE of the K-N tandem dimer demonstrated faster migration by the K-N tandem dimer (100 μM) after 500-nm light, an effect reversed by 400-nm light. The asterisk marks the location expected for tandem dimer migration, similar to tdTomato. Some cleavage of the tandem dimer to a monomer in this protein preparation was apparent. (F) Absorbance spectra of K-N tandem dimers confirm reversible photoswitching.
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Fig. 2. Control of photochromic FP domain association by light in cells. (A) Experimental plan for light-regulated interaction between Dronpa145N-CAAX (N-CAAX) and mNeptune-Dronpa145N (mNeptune-N). (B) Quantitation of membrane Dronpa fluorescence during 490/20-nm illumination. (C) 490/20-nm light induced off-photoswitching of Dronpa and loss of mNeptune from the plasma membrane. Scale bar, 20 μm. (D) Intensity profile between arrows in (C). (E) Experimental plan for light-regulated interaction between Dronpa145K-CAAX (K-CAAX) and mNeptune-N. (F) Quantitation of membrane Dronpa fluorescence during 490/20-nm illumination. (G) 490/20-nm light induced off-photoswitching of Dronpa and loss of mNeptune from the membrane. mNeptune reappeared at the membrane after 3-s on-photoswitching with 390/15-nm light. Scale bar, 20 μm. (H) Intensity profiles between arrows in (G).
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Fig. 3. A light-inducible single-chain GEF. (A) Proposed mechanism for photo-uncaging of N-I-N-CAAX activity. ITSN, intersectin. (B) Off-photoswitching of Dronpa fluorescence in N-I-N-CAAX versus 490/20-nm light dosage during microscopy. Whole-cell fluorescence results from five cells were quantified and normalized to the initial value. Error bars represent SD. (C) In NIH 3T3 cells expressing N-I-N-CAAX, 490/20-nm illumination for 30 s (off-switching) followed by incubation at 37°C for 30 min resulted in robust induction of filopodia, as revealed by mNeptune-Fascin. (D) Local illumination by 490/20-nm light locally induced filopodia, marked by mNeptune-fascin, in NIH 3T3 cells expressing N-I-N-CAAX. The dotted curves indicate the area of illumination. (E) Proposed mechanism for photo-uncaging of K-I-N-CAAX activity. (F) Off-photoswitching of Dronpa fluorescence in K-I-N-CAAX versus 490/20-nm light dosage during microscopy. The experiment was performed as in (B). (G) In NIH 3T3 cells expressing K-I-N-CAAX, exposure to 490/20-nm light for 30 s (off-switching) followed by incubation at 37°C for 30 min resulted in robust induction of filopodia. (H) Local illumination by 490/20-nm light locally induced filopodia, marked by mNeptune-fascin, in NIH 3T3 cells expressing K-I-N-CAAX. The dotted curves indicate the area of illumination. Scale bars in (C), (D), (G), and (H), 20 μm.
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Fig. 4. A light-inducible single-chain protease. (A) Strategy for sensing activity of the N-protease-N protein with mCherry-substrate-CAAX. (B) Distribution of mCherry in cells expressing mCherry-substrate-CAAX in the absence (left) or presence (middle) of cotransfected K-protease. The chart at right shows the fluorescence intensity profile along the line between the arrows in the images. (C) As expected from its size (81 kD), N-protease-N was excluded from the nucleus (left). 490/20-nm light for 15 s induced off-photoswitching of Dronpa fluorescence (Dronpa channel) and induced release of mCherry from the membrane (mCherry channel). The chart at right shows the intensity profile along the line between the arrows in the images, which confirmed that mCherry fluorescence decreases from the membrane and increases in the cytosol and nucleus after illumination. Scale bars in (B) and (C), 20 μm.
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