Tional adjustments take spot in nano- to microseconds (ns-ms), as in chain dynamics of disordered proteins, and protein folding in microseconds to minutes. Transitions along energetically unfavorable pathways can take up to hours or longer, as in protein misfolding (Borgia et al., 2011; Tosatto et al., 2015). (2013 Elsevier Ltd. All rights reserved. The figure was originally published as Figure 1 in Schuler and Hofmann, 2013. Current Opinion in Structural Biology, 23(1): 367. Further reproduction of this panel would will need permission from the copyright holder.) Bottom: (A) Picosecond (ps) to millisecond (ms) processes are normally examined with confocal techniques including polarization-resolved fluorescence lifetime measurements and Fluorescence Correlation Spectroscopy (FCS). Instance shown: chain dynamics of an IDP from nsFCS. (B) Conformational states are identified by person populations with characteristic positions in the FRET efficiency – lifetime diagrams as discussed within the sections Detection and characterization of intra-state dynamics and Future of smFRET (adapted from Soranno et al., 2012). (C) Speedy transitions measured making use of confocal microscopy could be analyzed employing the photon trajectory and applying a Chk2 web photon-by-photon maximum likelihood method (2018 Elsevier Ltd. All rights reserved. The figure was originally published as Figures 2 and 3 in Chung and Eaton, 2018. Current Opinion in Structural Biology, 48: 309. Further adaptation of this panel would will need permission in the copyright holder.) The timescale over which kinetics might be measured is usually extended for diffusing molecules at low concentrations by utilizing a recurrence analysis of CK1 Compound single particles (RASP, Hoffmann et al., 2011). (D) Non-equilibrium experiments over extended periods of time may be performed with microfluidic mixing devices. (Copyright 2011, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. Reproduced from Gambin et al., 2011, with permission. Nature Approaches eight:23941. Additional reproduction of this panel would want permission from the copyright holder.) (E) Slow changes in conformations more than a broad range of timescales can be followed in smFRET efficiency trajectories registered by single-photon counting (SPC) or cameras over minutes to several hours when the sample is immobilized (adapted from Figure 1 of Zosel et al., 2018). 2013, Elsevier Ltd. All rights reserved. Figure 3 (leading) and panel A was initially published as Figure 1 in Schuler and Hofmann, 2013. Additional reproduction of this panel would require permission from the copyright holder. 2018, Elsevier Ltd. All rights reserved. Panel C was initially published as Figures 2 and three in Chung and Eaton, 2018. Further adaptation of this panel would will need permission from the copyright holder. 2011, Nature Publishing Group, a division of Macmillan Publishers Restricted. All Rights Reserved. Panel D was originally published as Figure 1f in Gambin et al., 2011. Further reproduction of this panel would need permission from the copyright holder.Lerner, Barth, Hendrix, et al. eLife 2021;10:e60416. DOI: https://doi.org/10.7554/eLife.ten ofReview ArticleBiochemistry and Chemical Biology Structural Biology and Molecular BiophysicsSample preparation DyesFor studying biomolecular conformations and their dynamics with smFRET, the biomolecules of interest should be labeled with organic dyes which can be appropriate for single-molecule fluorescence detection (intrinsically fluorescent aromatic amino acids.
