title: Single molecule force spectroscopy reveals a weakly populated microstate of the FnIII domains of tenascin.
abstract: The native states of proteins exist as an ensemble of conformationally similar microstates. The fluctuations among different microstates are of great importance for the functions and structural stability of proteins. Here, we demonstrate that single molecule atomic force microscopy (AFM) can be used to directly probe the existence of multiple folded microstates. We used the AFM to repeatedly stretch and relax a recombinant tenascin fragment TNfnALL to allow the fibronectin type III (FnIII) domains to undergo repeated unfolding/refolding cycles. In addition to the native state, we discovered that some FnIII domains can refold from the unfolded state into a previously unrecognized microstate, N* state. This novel state is conformationally similar to the native state, but mechanically less stable. The native state unfolds at approximately 120 pN, while the N* state unfolds at approximately 50 pN. These two distinct populations of microstates constitute the ensemble of the folded states for some FnIII domains. An unfolded FnIII domain can fold into either one of the two microstates via two distinct folding routes. These results reveal the dynamic and heterogeneous picture of the folded ensemble for some FnIII domains of tenascin, which may carry important implications for the mechanical functions of tenascins in vivo.
authors.affiliation: Department of Chemistry, The University of British Columbia, Vancouver, BC, V6T 1Z1, Canada.
authors.forename: Yi
authors.initials: Y
authors.lastname: Cao
authors.forename: Hongbin
authors.initials: H
authors.lastname: Li
mesh_terms: D003198:Computer Simulation; D006801:Humans; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D009010:Monte Carlo Method; D010446:Peptide Fragments / Q000737:chemistry*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry*; D019063:Tenascin / Q000737:chemistry*
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D019063:Tenascin; C114731:tenascin fibronectin type III domain (1-92)