In Vitro-Evolved Peptides Bind Monomeric Actin and Mimic Actin-Binding Protein Thymosin-β4

Raphael J. Gübeli, Davide Bertoldo, Kenji Shimada, Christian B. Gerhold, Verena Hurst, Yuichiro Takahashi, Kai Harada, Ganesh K. Mothukuri, Jonas Wilbs, Masahiko Harata, Susan M. Gasser, Christian Heinis

    Research output: Contribution to journalArticlepeer-review

    2 Citations (Scopus)


    Actin is the most abundant protein in eukaryotic cells and is key to many cellular functions. The filamentous form of actin (F-actin) can be studied with help of natural products that specifically recognize it, as for example fluorophore-labeled probes of the bicyclic peptide phalloidin, but no synthetic probes exist for the monomeric form of actin (G-actin). Herein, we have panned a phage display library consisting of more than 10 billion bicyclic peptides against G-actin and isolated binders with low nanomolar affinity and greater than 1000-fold selectivity over F-actin. Sequence analysis revealed a strong similarity to a region of thymosin-β4, a protein that weakly binds G-actin, and competition binding experiments confirmed a common binding region at the cleft between actin subdomains 1 and 3. Together with F-actin-specific peptides that we also isolated, we evaluated the G-actin peptides as probes in pull-down, imaging, and competition binding experiments. While the F-actin peptides were applied successfully for capturing actin in cell lysates and for imaging, the G-actin peptides did not bind in the cellular context, most likely due to competition with thymosin-β4 or related endogenous proteins for the same binding site.

    Original languageEnglish
    Pages (from-to)820-828
    Number of pages9
    JournalACS Chemical Biology
    Issue number5
    Publication statusPublished - 2021 May 21

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Medicine


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