Angew. Chem. Int. Ed. 2013, 52, 7704-7708 Light-Regulated Stapled Peptides to Inhibit Protein-Protein Interactions Involved in Clathrin-Mediated Endocytosis.

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Angew. Chem. Int. Ed. 2013, 52, 7704-7708 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)

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, 3943-3946.

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

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

Photoswitchable Peptide Inhibitors

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

Photoisomerization and thermal-stability

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

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

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

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

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

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

Inhibition of CME in Living Cells

Transferrin Uptake Assay

Transferrin Uptake Assay

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

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.