Fluoromax 4 spectrophotometer

To assay nucleotide affinity, fluorescence intensity of 100 nM of BODIPY-FL-GDP or BODIPY-FL-GTP (Thermo Fisher Scientific) in 180 μl of assay buffer (30 mM HEPES, 100 mM KCl, 10 mM MgCl₂, pH 7.4) was measured using a Fluoromax-4 spectrophotometer (Horiba) (490 nm excitation, 509 nm emission). Yeast Met–tRNA_i was assayed in a similar manner using 20 nM BOP-N-Met–tRNA_i (tRNA probes) but with the addition of 1 mM GTP. Change in fluorescence intensity was measured upon addition of increasing amounts of apo–eIF2, incubating for 5 min at room temperature each time. Each measurement was blanked against a control without nucleotide to account for any affect of eIF2 and data were corrected for dilution effects caused by volume addition and normalized to starting values before being fitted to a single site binding model: y = 1 + [(ΔF_max - 1)*(x/(x + K_d))].

Fluorescent eIF2•BODIPY-GDP binary complex was formed by incubating apo–eIF2 with a 2× excess of BODIPY-FL-GDP (Thermo Fisher Scientific) for 20 min at room temperature. Excess nucleotide was removed by passing through a G-50 sephadex column (GE Healthcare). Labelling efficiency was calculated to exceed 90%. To measure GDP release, 20 nM eIF2•BODIPY-GDP was quickly mixed with 1 mM of unlabelled GDP (±eIF2B and ±GST-eIF5) in 180 μl of assay buffer (30 mM HEPES, 100 mM KCl, 10 mM MgCl₂, pH 7.4) and fluorescence intensity was continuously measured using a Fluoromax-4 spectrophotometer (Horiba) (490 nm excitation, 509 nm emission, 0.1 s integration time). Experimental data were fitted to exponential dissociation curves to determine the rate constants (K_off) at each eIF2B concentration. K_1/2 and K_max values were determined from curve fitting y = [(K_max × x)/(K_1/2 + x)] + c. Yeast eIF2B was recently shown to be a functional dimer (40 (link)), so likely interacts with eIF2 as a 2:2 complex. For consistency with prior studies, eIF2B and eIF2 concentrations stated and equations used refer to 5-subunit and 3-subunit monomeric complexes respectively.

The intrinsic fluorescence of albumin is mainly attributed to both tryptophan residues present in BSA molecule. The maximum emission of intrinsic fluorescence was determined for our albumin samples (1.5 mg/mL in PBS) from fluorescent emission spectra obtained with Horiba FluoroMax-4 spectrophotometer (250–500 nm range) under excitation wavelength at 270 nm (slit, 5 nm) [30 (link)]. The relative percent of quenching of tryptophan fluorescence was calculated using the following formula:
where Imax_Trp is maximal fluorescence intensity of albumin sample and Imax_Trp0 is the maximal fluorescence intensity of native non-glycated albumin (BSA).
For β-aggregation determination, thioflavin T (ThT) a specific fluorescent probe for amyloid cross β structure was used [29 (link)]. Albumin samples (2.5 µM) were incubated with 30 µM thioflavin T solution (dilution in H₂O) for 1 h at room temperature. The thioflavin emission spectra were obtained in the range of 250–600 nm under excitation at 435 nm (slit, 5 nm). The relative percent of β-amyloid formation was calculated using the following formula:
where Imax_ThT is maximal thioflavin T fluorescence intensity of albumin samples and Imax_ThT0 is the maximal thioflavin fluorescence intensity of nonglycated albumin (BSA).
All fluorescence spectra were corrected for the respective different absorption.

Steady-state photoluminescence spectra were acquired using a Fluoromax-4 spectrophotometer (HORIBA) with 370 nm photoexcitation from a Xe arc lamp. Transient photoluminescence decay measurements were performed by time-correlated single photon counting under a flow of N₂ using a Fluorolog-3 fluorescence lifetime spectrometer (HORIBA) with a 370 nm LED excitation source. The absolute PL quantum yields were determined under a flow of N₂ using a C9920 integrating sphere system (Hamamatsu Photonics). The method for determining the experimental k_RISC values is detailed in Supplementary Information Section 1.