Abstract
In this report, we have developed a simple approach using single-detector fluorescence autocorrelation spectroscopy (FCS) to investigate the Förster resonance energy transfer (FRET) of genetically encoded, freely diffusing crTC2.1 (mTurquoise2.1–linker–mCitrine) at the single molecule level. We hypothesize that the molecular brightness of the freely diffusing donor (mTurquoise2.1) in the presence of the acceptor (mCitrine) is lower than that of the donor alone due to FRET. To test this hypothesis, the fluorescence fluctuation signal and number of molecules of freely diffusing construct were measured using FCS to calculate the molecular brightness of the donor, excited at 405 nm and detected at 475/50 nm, in the presence and absence of the acceptor. Our results indicate that the molecular brightness of cleaved crTC2.1 in a buffer is larger than that of the intact counterpart under 405-nm excitation. The energy transfer efficiency at the single molecule level is larger and more spread in values as compared with the ensemble-averaging time-resolved fluorescence measurements. In contrast, the molecular brightness of the intact crTC2.1, under 488 nm excitation of the acceptor (531/40 nm detection), is the same or slightly larger than that of the cleaved counterpart. These FCS-FRET measurements on freely diffusing donor-acceptor pairs are independent of the precise time constants associated with autocorrelation curves due to the presence of potential photophysical processes. Ultimately, when used in living cells, the proposed approach would only require a low expression level of these genetically encoded constructs, helping to limit potential interference with the cell machinery.
Highlights
Förster resonance energy transfer (FRET) is a phenomenon in which energy is transferred, nonradiatively, from an excited donor (D) molecule to an acceptor (A) molecule that is in close proximity (≤10 nm) (Förster, 1948)
The statistical spreading of our molecular brightness and the estimated FRET efficiency could be attributed to the single-molecular processes or molecular conformations observed in fluorescence correlation spectroscopy (FCS) of freely diffusing crTC2.1 as compared with the traditional time-resolved fluorescence (Aplin et al, 2020)
As a proof of concept, we have demonstrated a simple approach for FRET analysis of freely diffusing crTC2.1 construct, at the single molecule level, using a traditional, single detector FCS setup
Summary
Förster resonance energy transfer (FRET) is a phenomenon in which energy is transferred, nonradiatively, from an excited donor (D) molecule to an acceptor (A) molecule that is in close proximity (≤10 nm) (Förster, 1948). Steady-state spectroscopy can be used to measure the energy transfer efficiency of FRET pairs based on the comparison of the time-averaged fluorescence intensity of the donor in the presence and absence of an acceptor (Lakowicz, 2006) This method has been applied to various mechanistic studies such as protein denaturation, enzyme-substrate binding, dye-DNA interaction, and RNA binding (Deniz et al, 2000; Zhuang et al, 2000; Elangovan et al, 2003; Ranjit et al, 2009; Zhang et al, 2013). The FRET analysis can be carried out on freely diffusing donor–linker–acceptor construct at the single molecule level in terms of the molecular brightness of the excited donor, in the presence and absence of an acceptor, using traditional, singledetector FCS setup where the fluorescence fluctuation and number of molecules can be measured. Assuming a three-dimensional (3D) Gaussian profile of the observation volume, the fluorescence fluctuation autocorrelation curve, GD(τ), due to translational diffusion and a fast photophysical process (with a time scale of τf ) can be written as following (Huang et al, 2002)
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