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. Author manuscript; available in PMC: 2017 Feb 4.
Published in final edited form as: Mol Cell. 2016 Feb 4;61(3):341–351. doi: 10.1016/j.molcel.2016.01.008

Figure 4. Co-translational folding of the NTD monitored in real-time by FRET.

Figure 4

Left panel, positions of the donor (BOF) and acceptor (BOP) dyes in the structure of gamma-B crystalline. Middle and right panels, Time-resolved folding of U (red) and H (blue) peptides monitored by FRET between BOP-Met at position 1 and BOF-Cys at position 88 in the stopped-flow apparatus. DA, both donor and acceptor dyes were present; A, control in the absence of the donor. Middle panel: direct comparison of FRET due to folding for the U and H variants. Right panel: FRET signal vs. acceptor direct excitation at donor wavelength; for better comparison, the traces with and without the donor for the U or H variants, respectively, were adjusted to the same starting level; the H traces are arbitrarily shifted from the U traces for visual clarity. Time courses were evaluated using a two-step model comprising a delay phase which shows no change in fluorescence and an exponential phase corresponding to a monomolecular folding reaction using GraphPad Prism. Delay times are 35 ± 0.3 s for H and 50 ± 0.5 s for U. The folding times determined by exponential fitting after delay are 39 ± 1 s for H and 59 ± 1 s for U. See also Figure S5.