(389b) Interfacial Concentration Dependence in Unfolding Proteins Using Afm

Filamin A is a long, high molecular mass (~280 kDa) acting-binding protein, constituting of 24 Ig domains in a monomeric form. Filamin A cross-links F-actin into three-dimensional isotropic resilient, stiffer, more solid-like networks. Dysfunction or deletion of human filamin causes various genetic disorder or deformations in melanoma cells due to reduced stiffness of cortical actin. Filamin's key role in actin organization, membrane stabilization, and the anchoring of transmembrane cell receptor proteins to the actin cytoskeleton, suggests that filamin has an important mechanical function. Here we show by atomic force microscopy (AFM) that all repeats for filamin extend and unfold at similar forces as observed and analyzed through sawtooth patterns. This novel AFM based nano-biotechnology opens a molecular mechanic approach for studying structure to function related properties of any type of individual biological macromolecules. In order to study forced unfolding of sub-domains within filamin, the minimal possible forces to unfold native filamin must be determined by eliminating effects of multiple chains or loops as much as possible. Here we show that force for unfolding individual domains of intact filamin increase with the increase in protein concentration adsorbed on the substrate (mica/gold) and thus results into high range of forces for unfolding as reported in previous studies. Similar interfacial concentrations dependent AFM induced unfolding experiments were performed and verified on titin-(I27)₈ peptide. Further domain unfolding forces for full length filamin monomer were compared with its sub-domain constructs- (R15-R16)_m ? consisting of repeats 15 and 16.