title: The mechanochemistry of copper reports on the directionality of unfolding in model cupredoxin proteins.
abstract: Understanding the directionality and sequence of protein unfolding is crucial to elucidate the underlying folding free energy landscape. An extra layer of complexity is added in metalloproteins, where a metal cofactor participates in the correct, functional fold of the protein. However, the precise mechanisms by which organometallic interactions are dynamically broken and reformed on (un)folding are largely unknown. Here we use single molecule force spectroscopy AFM combined with protein engineering and MD simulations to study the individual unfolding pathways of the blue-copper proteins azurin and plastocyanin. Using the nanomechanical properties of the native copper centre as a structurally embedded molecular reporter, we demonstrate that both proteins unfold via two independent, competing pathways. Our results provide experimental evidence of a novel kinetic partitioning scenario whereby the protein can stochastically unfold through two distinct main transition states placed at the N and C termini that dictate the direction in which unfolding occurs.
authors.affiliation: Department of Physics, King's College London, London WC2R 2LS, UK.
authors.forename: Amy E M
authors.initials: AEM
authors.lastname: Beedle
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, London WC2R 2LS, UK.
authors.forename: Ainhoa
authors.initials: A
authors.lastname: Lezamiz
authors.affiliation: CNRS - Institut de Biologie Physico-Chimique - PSL Research University, Laboratoire de Biochimie Théorique, 75005 Paris, France.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Stirnemann
authors.affiliation: Department of Physics, King's College London, London WC2R 2LS, UK.|Randall Division of Cell and Molecular Biophysics, King's College London, London WC2R 2LS, UK.
authors.forename: Sergi
authors.initials: S
authors.lastname: Garcia-Manyes
mesh_terms: D001400:Azurin / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli; D018625:Microscopy, Atomic Force; D056004:Molecular Dynamics Simulation; D036103:Nanotechnology; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D010970:Plastocyanin / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D013057:Spectrum Analysis
chemical_list: D001426:Bacterial Proteins; D010940:Plant Proteins; C050107:cupredoxin; D001400:Azurin; D003300:Copper; D010970:Plastocyanin