1. Angew. Chem. Int. Ed. 2013, 52,
Light-Regulated Stapled Peptides to Inhibit Protein-Protein Interactions Involved in Clathrin-Mediated Endocytosis
Laura Nevola, Andrés Martín-Quirós, Kay Eckelt, Núria Camarero, Sébastien Tosi, Artur Llobet, Ernest Giralt,* and Pau Gorostiza*
IRB Barcelona, IBEC, IDIBELL, University of Barcelona, CIBER-BBN, and ICREA (Spain)

2. Photocontrol of the secondary structure
of helical peptides by using azobenzenes
ex.) Photocontrol of the coiled-coil formation: Inhibition of DNA binding
F. Zhang et al., Angew. Chem. Int. Ed. 2010, 49,

3. Clathrin-Mediated Endocytosis (CME)
Plasma
membrane
Inside of
the cell
Clathrin
① Accumulation of clathrins
② Invagination
③ Neck formation
④ Isolation
Clathrin-coated pit (CCP) dynamics

4. Design of photoswitchable inhibitors
(BAP-long)
cysteine
(BSBCA)

5. Photoswitchable Peptide Inhibitors

6. Preparation of photoswitchable peptides
~HPLC~
(monitoring of the reaction)
(after purification)

7. Photoisomerization and thermal-stability

8. Fluorescence Polarization (FP) Binding Assay
(dark/500 nm irradiation)
(380 nm irradiation)

9. Fluorescence Polarization (FP) Binding Assay
(TL-1)
(TL-2)

10. Photoswitchable Peptide Inhibitors
(TL-1)
(TL-2)
(inactive)
(active)

11. Internalization of TLs
(FACS measurements)
(Fluorescence Imaging)
TLs are spontaneously internalized by cells
(does-dependent internalization)

12. TLs Toxicity Assay (MTT assay on HeLa cells)
(without irradiation)
(after irradiation)

13. Transferrin Uptake Assay
control
With TL-1
Red signals:
Alexa 647-transferrin
(Confocal)
10 mm
10 mm
1 mm
1 mm
(TIRFM)
(control)
(with TL-1)
Time-lapse

14. Inhibition of CME in Living Cells

15. Transferrin Uptake Assay

16. Transferrin Uptake Assay

17. Transferrin Uptake Assay
“dark” or
“under 500 nm”
“under 380 nm”
“stop”
“go”

18. Conclusions
Two cell-permeable molecules, TL (Traffic Light)-1 and TL-2, which are able to reversibly change the peptide conformation with external light stimuli, are prepared.
By using TL-1 and TL-2, the photoswitching of CME was demonstrated in the in-vitro as well as in living cells.