Figure 4: Surface immobilization strategy and correction factor retrieval for smFRET.
(a) Schematic showing the immobilisation of His-tagged protein to the surface coverslip for smFRET-TIRF experiments. The immobilized protein maintains its native conformation and activity as a result of the controlled orientation and inertness of the chemical surrounding provide by the Cu2+/PEG surface. (b) Solution measurements of emission spectra of L662BCNK.TC labelled with either donor FlAsH-EDT2 (DFM) or acceptor Tet1-TAMRA-X (AT) or both dyes (DFMAT) at the fixed excitation wavelength: 488 nm for DFM and DFAMTM and 561 nm for ATM (left). Emission spectra of the same samples, all at the fixed excitation wavelength of 488 nm are also shown (right) clearly demonstrating energy transfer from the donor to the acceptor (arrows). (c) Differentiation of donor-only, acceptor-only and donor-acceptor subpopulations by Alternating Laser EXitation (ALEX). A 2D-histogram/contour plot of the uncorrected stoichiometry (S) values versus the apparent FRET efficiency, known as proximity ratio (EPR), shows the position of each subpopulation (donor only, green; acceptor only, red; donor + acceptor, blue) determining the contributions of crosstalk of the donor emission into the acceptor detection channel and the direct excitation of the acceptor with the donor excitation laser. FRET efficiency (EPR) corrections, including accounting for background photons, are further discussed in Supplementary Material. (d) An example of a single Tet1-TAMRA-X acceptor dye photobleaching step, with corresponding increase in donor FlAsH-EDT2 emission. The photobleaching step manifests a drop in the calculated FRET efficiency (EPR). (e) An example of a corrected fluorescence intensity time trace of donor and acceptor (upper trace, donor FlAsH-EDT2 in green, acceptor Tet1-TAMRA-X in red) from which EPR (lower trace, blue) is calculated. See also Supplementary Figs S3 and S4.
