title: Single-Molecule Protein Folding Experiments Using High-Precision Optical Tweezers.
abstract: How proteins fold from linear chains of amino acids to delicate three-dimensional structures remains a fundamental biological problem. Single-molecule manipulation based on high-resolution optical tweezers (OT) provides a powerful approach to study protein folding with unprecedented spatiotemporal resolution. In this method, a single protein or protein complex is tethered between two beads confined in optical traps and pulled. Protein unfolding induced by the mechanical force is counteracted by the spontaneous folding of the protein, reaching a dynamic equilibrium at a characteristic force and rate. The transition is monitored by the accompanying extension change of the protein and used to derive conformations and energies of folding intermediates and their associated transition kinetics. Here, we provide general strategies and detailed protocols to study folding of proteins and protein complexes using optical tweezers, including sample preparation, DNA-protein conjugation and methods of data analysis to extract folding energies and rates from the single-molecule measurements.
authors.affiliation: Department of Cell Biology, School of Medicine and Integrated Graduate Program in Physical and Engineering Biology, Yale University, 333 Cedar Street, New Haven, CT, 06520, USA.
authors.forename: Junyi
authors.initials: J
authors.lastname: Jiao
authors.affiliation: Department of Cell Biology, School of Medicine and Integrated Graduate Program in Physical and Engineering Biology, Yale University, 333 Cedar Street, New Haven, CT, 06520, USA.
authors.forename: Aleksander A
authors.initials: AA
authors.lastname: Rebane
authors.affiliation: Department of Cell Biology, School of Medicine, Yale University, 333 Cedar Street, New Haven, CT, 06520, USA.
authors.forename: Lu
authors.initials: L
authors.lastname: Ma
authors.affiliation: Department of Cell Biology, School of Medicine, Yale University, 333 Cedar Street, New Haven, CT, 06520, USA. yongli.zhang@yale.edu.
authors.forename: Yongli
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D004268:DNA-Binding Proteins / Q000737:chemistry; D008390:Markov Chains; D044085:Microfluidics / Q000379:methods; D008958:Models, Molecular; D046912:Multiprotein Complexes; D052898:Optical Tweezers; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D050600:SNARE Proteins / Q000737:chemistry; D000072760:Single Molecule Imaging / Q000379:methods*
chemical_list: D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; D011506:Proteins; D050600:SNARE Proteins
keywords: Energy landscape; Hidden Markov modeling; Optical tweezers; Protein folding; SNARE assembly; SNARE proteins; Single-molecule manipulation; gp41