title: Dynamics of protein folding and cofactor binding monitored by single-molecule force spectroscopy.
abstract: Many proteins in living cells require cofactors to carry out their biological functions. To reach their functional states, these proteins need to fold into their unique three-dimensional structures in the presence of their cofactors. Two processes, folding of the protein and binding of cofactors, intermingle with each other, making the direct elucidation of the folding mechanism of proteins in the presence of cofactors challenging. Here we use single-molecule atomic force microscopy to directly monitor the folding and cofactor binding dynamics of an engineered metal-binding protein G6-53 at the single-molecule level. Using the mechanical stability of different conformers of G6-53 as sensitive probes, we directly identified different G6-53 conformers (unfolded, apo- and Ni(2+)-bound) populated along the folding pathway of G6-53 in the presence of its cofactor Ni(2+). By carrying out single-molecule atomic force microscopy refolding experiments, we monitored kinetic evolution processes of these different conformers. Our results suggested that the majority of G6-53 folds through a binding-after-folding mechanism, whereas a small fraction follows a binding-before-folding pathway. Our study opens an avenue to utilizing force spectroscopy techniques to probe the folding dynamics of proteins in the presence of cofactors at the single-molecule level, and we anticipated that this method can be used to study a wide variety of proteins requiring cofactors for their function.
authors: Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada.
authors: Yi
authors: Y
authors: Cao
authors: Hongbin
authors: H
authors: Li
mesh_terms: D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D009532:Nickel / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding
chemical_list: D001059:Apoproteins; D002352:Carrier Proteins; D009532:Nickel