Three-Color Single-Molecule FRET and Fluorescence Lifetime Analysis of Fast Protein Folding.

ABSTRACT: We describe the theory, experiment, and analysis of three-color Förster resonance energy transfer (FRET) spectroscopy for probing conformational dynamics of a fast-folding protein, α₃D. In three-color FRET, site-specific labeling of fluorophores is required to avoid ambiguity resulting from various species with different combinations of labeling positions. To this end, we first attached two dyes to a cysteine residue and an unnatural amino acid and then appended a cysteine residue to the C-terminus of the protein by the sortase-mediated ligation for attaching the third dye. To determine all three FRET efficiencies, we used alternating excitation of the donor and acceptor 1 with two picosecond-pulsed lasers. Since the folded and unfolded states are not distinguishable in binned f

SUBMITTER: Yoo J 

PROVIDER: S-EPMC6458097 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature