2.7. Single-Molecule Fluorescence Resonance Energy Transfer Measurement

The laser confocal fluorescence microscope MicroTime 200 (PicoQuant, Berlin, Germany) was used for single-molecule imaging, and a pulsed interleaved excitation (PIE) scheme [28] was used with two SPCM-AQRH detectors (Excelitas, Waltham, MA, USA) for recording fluorescence time traces at two different wavelengths. Two picosecond-pulsed diode laser heads (LDH-P-C-485 and LDH-P-C-640; PicoQuant, Berlin, Germany) were driven by a PDL 828 Sepia II driver (PicoQuant) at a repetition rate of 40 MHz, which allowed interleaved excitations for Alexa488 and Cy5. Each laser was coupled to the inverted microscope IX 73 (Olympus, Beijing, China) through a single-mode fiber, and was reflected by a dichroic mirror through a water-immersion objective (UPLSAPO 60×, N.A. 1.20). The protein sample was loaded onto a hybridization chamber (Thermo Fisher) glued to a glass coverslip (Thermo Fisher, Shanghai, China). The laser confocal point was set to about 50 μm above the coverslip. The excitation power at the back of the objective was about 100 μW for the 485 nm laser and about 35 μW for the 640 nm laser, as estimated with a power meter (PM20-FC; Thorlabs, Shanghai, China). Focused to a 100 μm pinhole, the fluorescence emission from the excited protein molecule was collected with the same objective. The donor and acceptor emissions were separated with a dichroic mirror (T635lpxr; Chroma, Xiamen, China). The donor emission was filtered with a 520/35 BP band pass, and the acceptor emission was filtered with a 690/70 BP band pass, before being focused onto the two SPCM-AQRH detectors.

The fluorescence outputs were recorded with a TimeHarp 260 PCI board (PicoQuant, Berlin, Germany) built into a PC workstation, and the data were stored in the time-tagged time-resolved module (PicoQuant, Berlin, Germany). The photon counts including f_Dex/Dem, f_Dex/Aem and f_Aex/Aem were obtained by binning the photons in 1 ms bins using SymPhoTime64 software (PicoQuant, Berlin, Germany). Here, f_Dex/Dem represents the photon count for the donor excitation and donor emission channel; f_Dex/Aem represents the photon count for the donor excitation and acceptor emission channel, and f_Aex/Aem represents the photon count for the acceptor excitation and acceptor emission channel. With all three fluorescence time traces recorded, the signals from the donor-only or acceptor-only protein molecules were filtered out, so as to ensure that an f_Dex/Aem photon only arises from a doubly labeled protein. A burst search was performed using a start/stop criterion as described [29,30].

The parameters for instrumentation and fluorophores were calibrated following the established protocol [29]. Double-stranded DNA oligonucleotides with different donor–acceptor distances, that is, the fluorophore conjugation sites separated by different numbers of bases (Figure S2), were used to determine the detection correction factor γ and the cross-talk terms (Figure S3). The cross-talk terms included the donor emission detected by the acceptor channel (donor leakage; abbreviated Lk) and the acceptor emission excited by the donor excitation wavelength (acceptor direct excitation; abbreviated Di). Alexa488 was conjugated to the C6-amino group of a dT nucleotide at the 5′ end of one oligonucleotide strand, whereas Cy5 was conjugated to the C6-amino group of an internal dT nucleotide of another oligonucleotide strand. The fluorophore-conjugated oligonucleotides were purchased from Sangong Biotech and further purified using a Source Q column. The purified single stranded DNA oligonucleotides were mixed at room temperature in a buffer containing 40 mM Tris-HCl (pH8.0) and 500 mM NaCl, heated to 95 °C for 2 min and gradually cooled down to room temperature in the dark for annealing. To ensure complete hybridization for the acceptor-labeled DNA strand, the donor-labeled strand had 50% molar excess (the donor-only double-stranded DNA could be filtered out using the PIE scheme). To prepare the donor-only and acceptor-only double-stranded DNA, a 10-fold excess of unlabeled DNA strand was used for annealing.

The smFRET measurements of phAimR were performed at 25 °C in 20 mM Tris-HCl (pH 7.5) buffer, containing 100 mM NaCl, 0.005% (vol/vol) Tween 20 (Thermo Fisher, Shanghai, China), 1 mM L-ascorbic acid and 1 mM methylviologen (Sigma-Aldrich, Shanghai, China). The concentration of the doubly labeled sample was about 100 pM. The smFRET data were collected for about 1 h. The threshold for photon count traces f_Dex/Dem, f_Dex/Aem and f_Aex/Aem was 3 to 7 counts per bin depending on the background dark counts. To be classified as a burst, the total photon counts (f_Dex/Dem + f_Dex/Aem) in the burst had to be at least 25 above the background threshold. The exact FRET efficiencies were calculated based on our calibrated parameters for the instrument and fluorophores, and the FRET efficiency distribution was analyzed with a multi-Gaussian mixture using our previously handwritten script [31].