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In Vitro-Evolved Peptides Mimic a Binding Motif of the G-Actin-Binding Protein Thymosin-B4 and Serve as Research Tools

preprint
submitted on 16.04.2020 and posted on 20.04.2020 by Raphael Gübeli, Davide Bertoldo, Kenji Shimada, Christian Gerhold, Verena Hurst, Yuichiro Takahashi, Jonas Wilbs, Masahiko Harata, Susan M. Gasser, Christian Heinis

Actin is the most abundant protein in eukaryotic cells and is key to many cellular functions. Natural products that specifically recognize the filamentous form of actin (F-actin) such as the bicyclic peptide phalloidin are important tools to study actin and are widely applied for imaging the cytoskeleton in cells. Herein, we aimed at developing peptide-based affinity reagents that selectively bind to the monomeric form of actin (G-actin), for which synthetic probes are not available. Panning a phage display library comprising more than a trillion different bicyclic peptides against G-actin yielded binders with low nanomolar affinity and greater than 1000-fold selectivity over F-actin. Sequence analysis revealed a strong similarity of the peptides' sequences to a region of thymosin-b4, a protein that weakly binds G-actin, and competition binding experiments confirmed a common binding region at the cleft between the subdomains 1 and 3 of actin. We tested the G-actin peptides as probes in pull-down and imaging experiments and applied a peptide variant with improved dissociation constant (Kd = 5 ± 2 nM) to measure the affinity of G-actin-binding natural product toxins.

Funding

Swiss National Science Foundation (Sinergia grant: 141945)

Human Frontiers Science Program (Grant: Actin and actin-related proteins – probing their nuclear function).

History

Email Address of Submitting Author

christian.heinis@epfl.ch

Institution

EPFL

Country

Switzerland

ORCID For Submitting Author

0000-0001-9982-9457

Declaration of Conflict of Interest

The authors have declared that no conflict of interest exists.

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