A mechanical unfolding intermediate in an actin-crosslinking protein

Ingo Schwaiger, Angelika Kardinal, Michael Schleicher, Angelika A Noegel, Matthias Rief
2003 Nature Structural & Molecular Biology  
In a moving cell, the cytoskeleton undergoes constant reorganization. The protein components of the cytoskeletal meshwork are therefore subject to mechanical deformation. Despite its obvious importance, we know little about the mechanical response of the individual cytoskeletal components to a force. Many actin crosslinking proteins share a common building scheme: two actin-binding domains are connected by a rod domain. The length and structure of the connecting rod varies considerably among
more » ... nsiderably among actin crosslinkers. The family of filamins has rod domains of the Ig fold. Depending on the specific filamin isoform, the number of rod domains can vary from 6 to 24 (ref. 1). Here we have used single-molecule mechanical experiments in combination with protein engineering to characterize the mechanical response to a stretching force of the rod domains of the Dictyostelium discoideum filamin ddFLN. ddFLN consists of an N-terminal actin-binding domain and a rod domain of six Ig domains (1-6, N terminus to C terminus; Fig. 1a) . Fucini et al. 2 and McCoy et al. 3 have shown that ddFLN dimerizes into an antiparallel homodimer via Ig domain 6. Here we explore the response of the ddFLN rod domains to a stretching force using singlemolecule force spectroscopy. RESULTS Mechanical stability of the ddFLN rod We investigated the stability of the dimer bond between each domain 6 of the two ddFLN monomers using a construct containing the entire dimerized rod lacking the N-terminal actin-binding domain (ddFLN1-6). The dimers were incubated on a freshly evaporated gold surface and picked up by an atomic force microscope (AFM) tip (Fig. 1b) . In force-extension curves obtained for this construct ( Fig. 1c) , similar to those of many other modular proteins investigated so far, a sawtooth pattern reflects the unfolding of individual Ig domains 4,5 . In the experiment, the molecules can be picked up anywhere along their contour, and so the number of unfolding peaks per force curve can vary from zero to the total number of domains in the rod. Many of the traces (see Fig. 1c ) exhibit clearly more than six unfolding peaks, which is the number of domains present in a ddFLN monomer. This result shows that under deformation forces the individual domains in the rod unfold before the dimer bond breaks. A lower estimate for the average strength of the dimer bond of ∼200 pN at pulling velocities between 100 nm s -1 and 1 µm s -1 can be obtained from the average force of the last rupture in our traces. This number is a lower estimate because we cannot distinguish rupture of the dimer bond from desorption of the molecule from either the tip or the substrate. Both events disrupt the mechanical connection between tip and substrate. Domain 4 unfolds via an intermediate state Most unfolding peaks show clear two-state unfolding of the Ig domains. The distance between two adjacent peaks is ∆L = 31.5 ± 0.1 nm (n = 636) and thus is consistent with the gain in length expected for the unfolding of a 100-residue domain. However, in the initial part of the force-extension curves, unfolding events with intermediate peaks are observed in the sawtooth pattern (green arrows, Fig. 1c ). This indicates that at least one of the six Ig domains does not unfold in a two-state manner but via an intermediate state. To identify the Ig domain with these special mechanical properties, we studied the domains individually. In force spectroscopy experiments, singlemolecule unfolding events must be distinguished from multiple 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 ∼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.
doi:10.1038/nsmb705 pmid:14718927 fatcat:qmplocrlrbcylbveexljqoz44m