Probing Actin Filament and Binding Protein Interaction Using an Atomic Force Microscopy

Abstract

Actin filaments play essential roles in many kinds of cellular functions by interacting with hundreds of actin binding proteins. Here we probe the interaction between actin filament and a binding protein, α-actinin, using an atomic force microscopy (AFM) and dynamic force spectroscopy (DFS). The distribution of rupturing events including specific and non-specific interactions of actin filament/α- actinin and BSA/α-actinin were analyzed. The rupture force of the actin filament/α-actinin binding was significantly larger than that of the BSA/α-actinin non-specific interaction, and the peaks represent typical multiple parallel bonds. In addition, based on the rupture forces in different loading rate DFS experiments, the dissociation constant of actin filament/α-actinin binding was estimated. The value is in good agreement with a previously reported value obtained by optical tweezer measurement. We expect that the present method will be useful for interaction measurement of actin filaments and many kinds of binding protein.

Characterization of Recognition Events between Proteins on a Single Molecule Level with Atomic Force Microscopy

“…These pairs include an anticancer peptide fragment of azurin (a bacterial protein that can be internalized in cancer cells and induce apoptosis) with p53, 28 p53 with Mdm2, 29 ribonuclease barnase with its inhibitor barstar, 30 and actin filament and binding protein. 31 Bonazza and co-workers also utilized AFM to investigate the complex formation between the von Willebrand factor (VWF) and factor VIII (FVIII), two essential hemostatic components of human blood. 32 Their results indicated that the fraction of VWF knots bound to FVIII was 25 ± 4%, and declined to 2 ± 1% when a complex was formed in the presence of the high salt buffer containing 500 mM Ca 2+ (Figure 3).…”

“…Moreover, the recognition events between ligand–receptor pairs have been investigated for understanding the physiological roles of these biological proteins at molecular level. These pairs include an anticancer peptide fragment of azurin (a bacterial protein that can be internalized in cancer cells and induce apoptosis) with p53, p53 with Mdm2, ribonuclease barnase with its inhibitor barstar, and actin filament and binding protein …”

Investigating the Effect of Tyrosine Kinase Inhibitors on the Interaction between Human Serum Albumin by Atomic Force Microscopy

“…There are many methods to study PPIs, such as the most commonly used immunoprecipitation (IP) [ 13 ], tandem affinity purification-mass spectrometry (TAP-MS) [ 14 ], and the pull-down experiment that is increasingly favored by researchers [ 15 ]. In addition, fluorescence resonance energy transfer (FRET), surface plasmon resonance (SPR), and optical tweezers are widely used in this research field [ 16 , 17 , 18 ]. For nanoscale protein assay experiments, AFM is a very powerful tool because it uses AFM tips modified with biomolecules to measure interactions with receptors.…”