Silvia Bellotto

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Personnes menant des recherches similaires (13)

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Generation of photoswitchable peptide ligands by phage display

Silvia Bellotto

Photoswitchable ligands are used to control and study complex biological systems such as folding of proteins and peptides, enzymatic reactions or neuronal signaling. They are typically developed by conjugating photochromic compounds to known ligands so that exposure to light results in the change of the binding affinity of the ligand. Many light-responsive ligands are based on azobenzene, a molecule that undergoes a pronounced change in geometry upon photoisomerization from trans to cis in picoseconds. Currently, photoswitchable ligands are available only to a limited number of targets and their development by rational design is complex. Furthermore, they show low affinity and a relatively small change in binding affinity between the cis and trans conformation. In my PhD work, I aimed at overcoming these shortcomings by establishing an in vitro evolution method to generate light-controlled peptide ligands to targets of choice. Combining the expertise on phage display technology of the Heinis' lab and the knowledge about azobenzene photoswitches of the Wegner's lab, I screened large combinatorial libraries of cyclic peptides containing an azobenzene-based linker for the generation of light controlled ligands. [...]

EPFL2015

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Phage Selection of Photoswitchable Peptide Ligands

Silvia Bellotto, Shiyu Chen, Christian Heinis, Inmaculada Rentero Rebollo, Hermann A. Wegner

Photoswitchable ligands are powerful tools to control biological processes at high spatial and temporal resolution. Unfortunately, such ligands exist only for a limited number of proteins and their development by rational design is not trivial. We have developed an in vitro evolution strategy to generate light-activatable peptide ligands to targets of choice. In brief, random peptides were encoded by phage display, chemically cyclized with an azobenzene linker, exposed to UV light to switch the azobenzene into cis conformation, and panned against the model target streptavidin. Isolated peptides shared strong consensus sequences, indicating target-specific binding. Several peptides bound with high affinity when cyclized with the azobenzene linker, and their affinity could be modulated by UV light. The presented method is robust and can be applied for the in vitro evolution of photoswitchable ligands to virtually any target.

American Chemical Society2014

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The measurement of activities from individual proteases in biological samples is difficult because of the numerous proteases, their overlapping activities, and the lack of specific substrates. We applied selective protease inhibitors based on bicyclic peptides (>2000-fold selective over related proteases) to block individual proteases, allowing the quantification of their net activities. In protease mixtures, activity contributions of the serine proteases plasma kallikrein and urokinase-type plasminogen activator (uPA) were accurately quantified. In a tumor extract, we could quantify uPA activity. Because bicyclic peptide inhibitors toward virtually any protease can be generated by phage display, the approach should be applicable to any protease.

2012

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