Direct Observation of the Reversible Two‐State Unfolding and Refolding of an α/β Protein by Single‐Molecule Atomic Force Microscopy |
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| Authors: | Chengzhi He Prof. Dr. Chunguang Hu Prof. Dr. Xiaodong Hu Prof. Dr. Xiaotang Hu Adam Xiao Prof. Dr. Thomas T. Perkins Prof. Dr. Hongbin Li |
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| Affiliation: | 1. Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1 (Canada);2. State Key Laboratory of Precision Measurements Technology and Instruments, School of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin, 300072 (China);3. JILA, NIST and University of Colorado Boulder, Dept. of Molecular, Cellular, and Developmental Biology, University of Colorado, 440 UCB Boulder, CO 80309 (USA) |
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| 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. |
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| Keywords: | atomic force microscopy fluctuation theorem force spectroscopy protein folding single‐molecule studies |
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