Fig. 2: Probing conformational changes of biomolecules by multiwell plate smFRET.

a The multiwell plate format provides the convenience to screen multiple sample repeats or concentration gradients. b Evaluation of the accuracy and precision of multiwell plate smFRET measurements using rigid DNA ruler constructs. 96 independent, but identical repeats of a mixture of two double-stranded DNA ruler constructs with 21- and 9-bp spacing between the acceptor (red) and donor (green) fluorophores. c Cumulated FRET efficiency histogram of all 96 wells (top) and the two-dimensional (2-D) histogram of E_FRET versus multiwell plate repeat (bottom). The error bars indicate the standard deviation (SD) derived from the 96 measurement repeats. Expected E_FRET for the two constructs (calculated by accessible volume (AV) simulations³⁸), are indicated as black solid lines. The confidence intervals (black dashed lines) are derived from the uncertainty of the Förster radius. d 2-D E_FRET histogram of the salt-dependent structural dynamics of the hpT₅ DNA hairpin in a 96-well plate. Inset: Schematic of the hairpin structure with donor and acceptor fluorophore positions indicated. e E_FRET histogram of hpT₅ at 782 mM NaCl fitted with a dynamic 3-Gaussian model^5,40,41 (red line and black dashed lines). The intermediate E_FRET population originates from molecules changing their conformation between open and closed state during the diffusion through the confocal volume. f Opening rates of hpT₅ decrease linearly with increasing NaCl concentration (red straight line). Closing rates of hpT₅ exhibit a non-linear behavior at lower salt concentrations, which is well described by a model considering the apparent concentration (red curved line). Data are presented as predicted value +/−68% confidence interval (CI) as derived by the dynamic 3-G fit. g 2-D E_FRET histogram of GdmCl-induced unfolding of the protein S6 in a 96-well plate format. Inset: Schematic of the protein with donor and acceptor fluorophore positions indicated. h FRET efficiency histogram of S6 at 3.2 M GdmCl (gray bars) and the double Gaussian fit (red line) to quantify the fraction of unfolded molecules. i Fraction of unfolded S6 molecules as a function of GdmCl concentration. Data are presented as predicted value +/−68% CI as derived by the static 2-G fit. Data was fitted using Eq. 11 (red line). Source data are provided as a Source data file.