Complexity of the Folding Transition of the B Domain of Protein A Revealed by the High-Speed Tracking of Single-Molecule Fluorescence Time Series

• DOI: 10.1021/acs.jpcb.5b00414
• Publication Date: 2015-05-04T13:11:05Z
• Authors: Hiroyuki Oikawa; Kiyoto Kamagata; Munehito Arai; Satoshi Takahashi
• Keywords: Equilibrium unfolding; Single-molecule FRET; Folding (DSP implementation); Acceptor

The equilibrium unfolding transition of the B domain protein A (BdpA) was investigated by using single-molecule fluorescence spectroscopy based on line-confocal detection fast-flowing samples. method achieved time resolution 120 μs and observation a few milliseconds in time-series measurements resonance energy transfer (FRET). Two samples BdpA doubly labeled with donor acceptor fluorophores, first possessing fluorophores at residues 22 55 (sample 1) second 5 2), were prepared. induced guanidium chloride (GdmCl) monitored bulk demonstrated that both obey apparent two-state unfolding. In absence GdmCl, FRET for showed single peak assignable to native state (N). efficiency N shifts lower values as increase GdmCl concentration, suggesting swelling structure. At higher concentration convert unfolded (U). Near midpoint sample 1, kinetic exchange between U causes averaging two states relative fluctuation. series different molecules slightly efficiencies, heterogeneity. Thus, high-speed tracking signals from revealed complexity heterogeneity hidden behavior folding.