title: Reversible Unfolding and Folding of the Metalloprotein Ferredoxin Revealed by Single-Molecule Atomic Force Microscopy.
abstract: Plant type [2Fe-2S] ferredoxins function primarily as electron transfer proteins in photosynthesis. Studying the unfolding-folding of ferredoxins in vitro is challenging, because the unfolding of ferredoxin is often irreversible due to the loss or disintegration of the iron-sulfur cluster. Additionally, the in vivo folding of holo-ferredoxin requires ferredoxin biogenesis proteins. Here, we employed atomic force microscopy-based single-molecule force microscopy and protein engineering techniques to directly study the mechanical unfolding and refolding of a plant type [2Fe-2S] ferredoxin from cyanobacteria Anabaena. Our results indicate that upon stretching, ferredoxin unfolds in a three-state mechanism. The first step is the unfolding of the protein sequence that is outside and not sequestered by the [2Fe-2S] center, and the second one relates to the force-induced rupture of the [2Fe-2S] metal center and subsequent unraveling of the protein structure shielded by the [2Fe-2S] center. During repeated stretching and relaxation of a single polyprotein, we observed that the completely unfolded ferredoxin can refold to its native holo-form with a fully reconstituted [2Fe-2S] center. These results demonstrate that the unfolding-refolding of individual ferredoxin is reversible at the single-molecule level, enabling new avenues of studying both folding-unfolding mechanisms, as well as the reactivity of the metal center of metalloproteins in vitro.
authors.affiliation: State Key Laboratory of Precision Measurements Technology and Instruments, School of Precision Instrument and Optoelectronics Engineering, Tianjin University , Tianjin 300072, People's Republic of China.|Department of Chemistry, University of British Columbia , Vancouver, British Columbia V6T 1Z1, Canada.
authors.forename: Hai
authors.initials: H
authors.lastname: Lei
authors.affiliation: Department of Chemistry, University of British Columbia , Vancouver, British Columbia V6T 1Z1, Canada.
authors.forename: Yabin
authors.initials: Y
authors.lastname: Guo
authors.affiliation: State Key Laboratory of Precision Measurements Technology and Instruments, School of Precision Instrument and Optoelectronics Engineering, Tianjin University , Tianjin 300072, People's Republic of 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, People's Republic of 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, People's Republic of China.
authors.forename: Xiaotang
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, People's Republic of China.|Department of Chemistry, University of British Columbia , Vancouver, British Columbia V6T 1Z1, Canada.
authors.forename: Hongbin
authors.identifier: 0000-0001-7813-1332
authors.initials: H
authors.lastname: Li
mesh_terms: D017033:Anabaena / Q000737:chemistry*; D005288:Ferredoxins / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D015202:Protein Engineering; D017510:Protein Folding
chemical_list: D005288:Ferredoxins