Temperature-cycle single-molecule FRET microscopy on polyprolines

Abstract

Accessing the microsecond dynamics of a single fluorescent molecule in real time is difficult because molecular fluorescence rates usually limit the time resolution to milliseconds. We propose to apply single-molecule temperature-cycle microscopy to probe molecular dynamics at microsecond timescales. Here, we follow donor and acceptor signals of single FRET-labeled polyprolines in glycerol to investigate their conformational dynamics. We observe a steady-state FRET efficiency distribution which differs from theoretical distributions for isotropically orientated fluorescent labels. This may indicate that the orientation of fluorescent labels in glycerol is not isotropic and may reflect the influence of the dye linkers. With proper temperature-cycle parameters, we observed large FRET changes in long series of cycles of the same molecule. We attribute the main conformational changes to reorientations of the fluorescent labels with respect to the oligopeptide chain, which take place in less than a few microseconds at the highest temperature of the cycle (250 K). We were able to follow the FRET efficiency of a particular construct for more than 2000 cycles. This trajectory displays switching between two conformations, which give rise to maxima in the FRET efficiency histogram. Our experiments open the possibility to study biomolecular dynamics at a time scale of a few microseconds at the single-molecule level.

The Impact of Heterogeneity and Dark Acceptor States on FRET: Implications for Using Fluorescent Protein Donors and Acceptors

“…The average efficiency 〈E〉 for the dynamic regime population, whose distribution is actually slightly asymmetric, is 0.50 with a standard deviation of 0.01, while the static isotropic regime population is characterized by a very broad asymmetric distribution with 〈E〉 = 0.38 and a very large standard deviation of 0.25 reflecting that. This distribution was similar to probability density distributions used to analyze expected inter-dye distances for polyprolines in glycerol [42] based on calculations presented by Dale and colleagues [25].…”

Temperature-cycle microscopy reveals single-molecule conformational heterogeneity

“…Furthermore, we observed reversible transitions between a high 4 FRET value and a low FRET value on individual FRET constructs by applying temperature cycles between 170 K and 250 K 26 . Furthermore, we observed reversible transitions between a high 4 FRET value and a low FRET value on individual FRET constructs by applying temperature cycles between 170 K and 250 K 26 .…”

“…The heating power was set to reach a higher temperature of 290 K in a temperature cycle according to our previous calibration 22,26 . In each temperature cycle, the heating beam was applied for 20 µs and the 50 MHz-pulsed excitation beam was applied for 500 ms or CW excitation was applied for 60 ms. That is probably due to their low donor fluorescence intensities.…”

“…This may be due to the different temperature-cycle parameters applied during measurements 26 . This may be due to the different temperature-cycle parameters applied during measurements 26 .…”

“…A dynamical process can thus be studied as a consecutive series of snapshots of frozen states with controllable time steps. In previous work, we demonstrated temperature-cycle microscopy on FRET labeled all-trans polyprolines in glycerol 26 . A schematic view of the temperature-cycle method is shown in Fig.…”