title: Direct Observation of the Reversible Two-State Unfolding and Refolding of an α/β Protein by Single-Molecule Atomic Force Microscopy.
abstract: Directly observing protein folding in real time using atomic force microscopy (AFM) is challenging. Here the use of AFM to directly monitor the folding of an α/β protein, NuG2, by using low-drift AFM cantilevers is demonstrated. At slow pulling speeds (<50 nm s(-1)), the refolding of NuG2 can be clearly observed. Lowering the pulling speed reduces the difference between the unfolding and refolding forces, bringing the non-equilibrium unfolding-refolding reactions towards equilibrium. At very low pulling speeds (ca. 2 nm s(-1)), unfolding and refolding were observed to occur in near equilibrium. Based on the Crooks fluctuation theorem, we then measured the equilibrium free energy change between folded and unfolded states of NuG2. The improved long-term stability of AFM achieved using gold-free cantilevers allows folding-unfolding reactions of α/β proteins to be directly monitored near equilibrium, opening the avenue towards probing the folding reactions of other mechanically important α/β and all-β elastomeric proteins.
authors.affiliation: Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1 (Canada).
authors.forename: Chengzhi
authors.initials: C
authors.lastname: He
authors.affiliation: State Key Laboratory of Precision Measurements Technology and Instruments, School of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin, 300072 (China).
authors.forename: Chunguang
authors.initials: C
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Precision Measurements Technology and Instruments, School of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin, 300072 (China).
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Precision Measurements Technology and Instruments, School of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin, 300072 (China).
authors.forename: Xiaotang
authors.initials: X
authors.lastname: Hu
authors.affiliation: Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1 (Canada).
authors.forename: Adam
authors.initials: A
authors.lastname: Xiao
authors.affiliation: JILA, NIST and University of Colorado Boulder, Dept. of Molecular, Cellular, and Developmental Biology, University of Colorado, 440 UCB Boulder, CO 80309 (USA).
authors.forename: Thomas T
authors.initials: TT
authors.lastname: Perkins
authors.affiliation: Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1 (Canada). Hongbin@chem.ubc.ca.|State Key Laboratory of Precision Measurements Technology and Instruments, School of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin, 300072 (China). Hongbin@chem.ubc.ca.
authors.forename: Hongbin
authors.initials: H
authors.lastname: Li
mesh_terms: D018625:Microscopy, Atomic Force; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D011506:Proteins / Q000737:chemistry*
chemical_list: D011506:Proteins
keywords: atomic force microscopy; fluctuation theorem; force spectroscopy; protein folding; single-molecule studies