title: Mechanical Folding and Unfolding of Protein Barnase at the Single-Molecule Level.
abstract: The unfolding and folding of protein barnase has been extensively investigated in bulk conditions under the effect of denaturant and temperature. These experiments provided information about structural and kinetic features of both the native and the unfolded states of the protein, and debates about the possible existence of an intermediate state in the folding pathway have arisen. Here, we investigate the folding/unfolding reaction of protein barnase under the action of mechanical force at the single-molecule level using optical tweezers. We measure unfolding and folding force-dependent kinetic rates from pulling and passive experiments, respectively, and using Kramers-based theories (e.g., Bell-Evans and Dudko-Hummer-Szabo models), we extract the position of the transition state and the height of the kinetic barrier mediating unfolding and folding transitions, finding good agreement with previous bulk measurements. Measurements of the force-dependent kinetic barrier using the continuous effective barrier analysis show that protein barnase verifies the Leffler-Hammond postulate under applied force and allow us to extract its free energy of folding, ΔG0. The estimated value of ΔG0 is in agreement with our predictions obtained using fluctuation relations and previous bulk studies. To address the possible existence of an intermediate state on the folding pathway, we measure the power spectrum of force fluctuations at high temporal resolution (50 kHz) when the protein is either folded or unfolded and, additionally, we study the folding transition-path time at different forces. The finite bandwidth of our experimental setup sets the lifetime of potential intermediate states upon barnase folding/unfolding in the submillisecond timescale.
authors.affiliation: Small Biosystems Lab, Departament de Física Fonamental, Universitat de Barcelona, Barcelona, Spain.
authors.forename: Anna
authors.initials: A
authors.lastname: Alemany
authors.affiliation: Small Biosystems Lab, Departament de Física Fonamental, Universitat de Barcelona, Barcelona, Spain.
authors.forename: Blanca
authors.initials: B
authors.lastname: Rey-Serra
authors.affiliation: Small Biosystems Lab, Departament de Física Fonamental, Universitat de Barcelona, Barcelona, Spain; Networking Research Center of Bioengineering, Biomaterials and Nanomedicine, Instituto Carlos III, Madrid, Spain.
authors.forename: Silvia
authors.initials: S
authors.lastname: Frutos
authors.affiliation: Department of Physics, Informatics and Mathematics, University of Modena and Reggio Emilia, Modena, Italy; Consiglio Nazionale delle Ricerche Institute of Nanosciences S3, Modena, Italy.
authors.forename: Ciro
authors.initials: C
authors.lastname: Cecconi
authors.affiliation: Small Biosystems Lab, Departament de Física Fonamental, Universitat de Barcelona, Barcelona, Spain; Networking Research Center of Bioengineering, Biomaterials and Nanomedicine, Instituto Carlos III, Madrid, Spain. Electronic address: fritort@gmail.com.
authors.forename: Felix
authors.initials: F
authors.lastname: Ritort
mesh_terms: D001426:Bacterial Proteins; D001696:Biomechanical Phenomena; D004548:Elasticity; D007700:Kinetics; D055595:Mechanical Phenomena; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D058767:Protein Unfolding; D012260:Ribonucleases / Q000737:chemistry*; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D010455:Peptides; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease