1. In Vitro -Evolved Peptides Bind Monomeric Actin and Mimic Actin-Binding Protein Thymosin-β4.
- Author
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Gübeli RJ, Bertoldo D, Shimada K, Gerhold CB, Hurst V, Takahashi Y, Harada K, Mothukuri GK, Wilbs J, Harata M, Gasser SM, and Heinis C
- Subjects
- Actin Cytoskeleton chemistry, Binding Sites, Binding, Competitive, HeLa Cells, Humans, In Vitro Techniques, Marine Toxins chemistry, Oxazoles chemistry, Peptide Library, Protein Binding, Protein Conformation, Structure-Activity Relationship, Actins chemistry, Microfilament Proteins chemistry, Peptides, Cyclic chemistry, Thymosin chemistry
- Abstract
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.
- Published
- 2021
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