Complexity of the folding transition of the b domain of protein a revealed by the high-speed tracking of single-molecule fluorescence time series

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Abstract

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

Original languageEnglish
Pages (from-to)6081-6091
Number of pages11
JournalJournal of Physical Chemistry B
Volume119
Issue number20
DOIs
Publication statusPublished - 2015 May 21

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Surfaces, Coatings and Films
  • Materials Chemistry

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