Jobin yvon fluorolog

Fluorescence spectra of SecY**EG PLs were measured on a Jobin Yvon Fluorolog (Horiba Scientific), exciting at 493 nm and measuring emission spectra. Measurements were performed in TKM buffer (20 mM Tris pH 7.5, 50 mM KCl, 2 mM MgCl₂), starting with 50 nM SecY**EG, then adding sequentially 1 µM nucleotide-stripped SecA, 1 mM AMPPNP, ADP or ATP, and 0.7 µM pOA. At least 5 mins were left after each addition before measuring, and each spectrum was repeated three times to confirm that a steady state was reached. Data were corrected for dilution before plotting.
FRET measurements for SecY**EG in DDM were performed in TSG buffer (20 mM Tris pH 7.5, 130 mM NaCl, 10% glycerol) with 0.02% DDM and 2 mM MgCl₂ on a Nanodrop 3300 Fluorospectrometer (Thermo Scientific, Waltham MA, ). 100 nM DDM-solubilised SecY**EG was measured alone, with 1 µM SecA, with SecA and 1 mM ADP, or with SecA and 1 mM AMPPNP. After excitation with blue light, fluorescence signals at 519 nm (donor; F_D) and 617 nm (acceptor; F_A) were measured, and FRET efficiencies calculated as F_A / (F_D + F_A). For each data point, three repeats were taken and the average FRET efficiency used.

Hexachlorofluorescein-labelled (HEX) DNA and RNA oligonucleotide probes (Supplementary Table S1) were chosen based on previous work (19 (link)) (Eurofins Genomics) and diluted to 5 nM in assay buffer (150 mM NaCl, 25 mM HEPES pH 7.45, 1 mM TCEP). An assay volume of 150 μl was dispensed into a Hellma 10 × 2 mm Suprasil quartz cuvette (Merck #Z802778). For double-stranded substrates, complementary oligonucleotides were mixed at a 1:1 molar ratio and heated to 95°C prior to slowly cooling to room temperature overnight in an insulated chamber. Proteins were then titrated into the cuvette at increasing concentrations and fluorescence anisotropy measurements were recorded at 20°C with a Jobin Yvon Fluorolog (Horiba Scientific) with excitation and emission wavelengths of 530 and 550 nm, respectively. Measurements were taken with an integration time of 0.5 s and averaged across four accumulations. Dilutions and titrations were carried out in triplicate and averages with standard deviations were plotted before fitting the data (GraphPad Prism) with a single-component binding equation  to determine the dissociation binding constant (K_D) where B_max is the maximum anisotropy change (saturated anisotropy value – starting value), X is the concentration of protein, and C is the anisotropy value at which the X axis value is 0.