Single-molecule photon stamping FRET spectroscopy study of enzymatic conformational dynamics

Abstract

The fluorescence resonant energy transfer (FRET) from a donor to an acceptor via transition dipole-dipole interactions decreases the donor's fluorescent lifetime. The donor's fluorescent lifetime decreases as the FRET efficiency increases, following the equation: E(FRET) = 1 - τ(DA)/τ(D), where τ(D) and τ(DA) are the donor fluorescence lifetime without FRET and with FRET. Accordingly, the FRET time trajectories associated with single-molecule conformational dynamics can be recorded by measuring the donor's lifetime fluctuations. In this article, we report our work on the use of a Cy3/Cy5-labeled enzyme, HPPK to demonstrate probing single-molecule conformational dynamics in an enzymatic reaction by measuring single-molecule FRET donor lifetime time trajectories. Compared with single-molecule fluorescence intensity-based FRET measurements, single-molecule lifetime-based FRET measurements are independent of fluorescence intensity. The latter has an advantage in terms of eliminating the analysis background noise from the acceptor fluorescence detection leak through noise, excitation light intensity noise, or light scattering noise due to local environmental factors, for example, in a AFM-tip correlated single-molecule FRET measurements. Furthermore, lifetime-based FRET also supports simultaneous single-molecule fluorescence anisotropy.

Fig. 1

(A) Crystal structure of HPPK. The cyan spirals represent the α helices and the yellow arrows are the β strands. The loops are shown by the red pipes. FRET dyes pair of Cy3–Cy5 are labeled on residue 88 and residue 142, respectively. (B) The reaction of HPPK-catalyzed pyrophosphoryl transfer from HP to HPPP.

Fig. 2

Photon stamping concept and definition of the parameters: (A) scheme of a train of laser excitation pulses and detected emission photons; (B) scheme of the time-stamped photon sequence. The delay time τ_p is the time delay between the photoexciation event and the photon emission; the real time t is the chronic time of detecting emission photons; for each detected photon, both τ_p and t_p are simultaneously recorded.

Fig. 3

Single-molecule photon-stamping measurement and data analysis. All the data are collected from a Cy3–Cy5 labeled single HPPK molecule under the enzymatic reaction conditions: 100 μM ATP and 100 μM HP. (A) An example of the single-molecule photon-stamping raw data from the donor channel in a 0.8 second period (5.8–6.6 s). Each data dot corresponds to a detected photon plotted by the delay time (τ_p) vs. its chronic arrival time (t). (B) Histograms of the delay times of the photons in a 10 ms period from the trajectory shown in A. The left panel is histogram of delay times in 10 ms (6.08–6.09 s), corresponding to the low energy transfer efficiency from donor to acceptor. The right panel is histogram of delay times in 10 ms (6.30–6.31 s), corresponding to the high energy transfer efficiency. (C) Lifetime trajectory of donor (τ_DA) calculated from the trajectory in A with 10 ms binning. The arrows show the positions (6.08–6.09 s) and (6.30–6.31 s) of the lifetime trajectory. (D) Intensity trajectory of donor calculated from trajectory in A with 10 ms binning.

Fig. 4

Single-molecule fluorescence images (10 μm × 10 μm) of Cy3 (A) and Cy5 (B) labeled HPPK molecules in 1% agarose gel Tris-HCl buffer solution (pH = 8.3). (C) Single-molecule fluorescence intensity trajectory of donor from Cy3/Cy5-labeled HPPK under the enzymatic reaction condition with 100 μM ATP and 100 μM HP. (D) Lifetime trajectory of Cy3, τ_DA (purple) from Cy3/Cy5-labeled HPPK and Lifetime trajectory of Cy3, τ_D (green) with only Cy3 labeled under the enzymatic reaction condition with 100 μM ATP and 100 μM HP. (E) E_FRET trajectory of Cy3–Cy5 labeled HPPK, calculated from lifetime time trajectory in D, where we use the fitting value of 2.65 ns as τ_D. (F, G, H) Auto-correlations from the intensity trajectory in C, lifetime trajectory τ_DA in D, and E_FRET trajectory in E show the same decay time τ = 0.14 ± 0.01 s.

Fig. 5

(A) A fluorescence intensity-time trajectory of donor (green) and acceptor (red) in a single-molecule AFM-FRET measurement on one HPPK (labeled Cy3–Cy5 on 88c, 142c), the experiment was performed in 50 mM tris buffer (pH = 8.3) and 10 mM MgCl₂, but no enzymatic reaction substrate were added. The donor–acceptor intensities change show the strong correlation with the AFM tip approaching-withdrawing movements. (B) Intensity-based FRET efficiency-time trajectory calculated from trajectory A, and the E_FRET changes obviously in correlation with the AFM tip approaching-withdrawing movements, which is an artifact background that complicating the real protein dynamics analysis. (C) Lifetime-based FRET efficiency-time trajectory from the lifetime measurement, the lifetime-based E_FRET shows no significant correlation with the AFM tip approaching-withdrawing movements, effectively removed the AFM-FRET measurement artifact background.