A mechanical unfolding intermediate in an actin-crosslinking protein

• Published in: Nature structural & molecular biology Vol. 11; no. 1; pp. 81 - 85
• Main Authors: Rief, Matthias, Schwaiger, Ingo, Kardinal, Angelika, Schleicher, Michael, Noegel, Angelika A
• Format: Journal Article
• Language: English
• Published: United States Nature Publishing Group 01.01.2004
• Subjects: Actin; Actins - metabolism; Animals; Contractile Proteins - chemistry; Contractile Proteins - genetics; Contractile Proteins - metabolism; Cross-Linking Reagents; Crosslinking; Dictyostelium - genetics; Dictyostelium - metabolism; Dictyostelium discoideum; Dimerization; filamin; Filamins; In Vitro Techniques; Microfilament Proteins - chemistry; Microfilament Proteins - genetics; Microfilament Proteins - metabolism; Microscopy, Atomic Force; Mutagenesis, Site-Directed; Physiological aspects; Protein Denaturation; Protein Folding; Protein Structure, Tertiary; Proteins; Protozoan Proteins - chemistry; Protozoan Proteins - genetics; Protozoan Proteins - metabolism; Germany
• ISSN: 1545-9993; 1545-9985
• DOI: 10.1038/nsmb705

Many F-actin crosslinking proteins consist of two actin-binding domains separated by a rod domain that can vary considerably in length and structure. In this study, we used single-molecule force spectroscopy to investigate the mechanics of the immunoglobulin (Ig) rod domains of filamin from Dictyostelium discoideum (ddFLN). We find that one of the six Ig domains unfolds at lower forces than do those of all other domains and exhibits a stable unfolding intermediate on its mechanical unfolding pathway. Amino acid inserts into various loops of this domain lead to contour length changes in the single-molecule unfolding pattern. These changes allowed us to map the stable core of approximately 60 amino acids that constitutes the unfolding intermediate. Fast refolding in combination with low unfolding forces suggest a potential in vivo role for this domain as a mechanically extensible element within the ddFLN rod.