In Vitro-Evolved Peptides Mimic a Binding Motif of the G-Actin-Binding Protein Thymosin-B4 and Serve as Research Tools
Preprints are manuscripts made publicly available before they have been submitted for formal peer review and publication. They might contain new research findings or data. Preprints can be a draft or final version of an author's research but must not have been accepted for publication at the time of submission.
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.