Raw membrane images from in vitro deoxyhypusination assays and numerical data for MST measurements presented in publication 10.1016/j.celrep.2024.114831

Microscale thermophoresis (MST) All the DHPS variants were labeled on surface-exposed cysteines with fluorophore-tagged maleimide according to manufacturer protocol (NanoTemper Technologies), and the labeled protein was eluted to MST buffer (30 mM HEPES pH 8.0, 150 mM NaCl, 0.05% Tween 20). The degree-of-labelling was verified to be between 0.5 and 1 with absorbance measurements at 280 nm and 650 nm according to manufacturer protocol. ERK2 was used in a 6xHis-tagged form and was buffer exchanged to MST buffer by 3 rounds of dilution and concentration in Amicon Ultra Centrifugal Filter (10kDa MWCO; Merck). ERK2 variants, were used at a concentration achieved after the last round of ultrafiltration (>250 μM in assay), while for studies of DHPS mutants, the highest ERK2 concentration in assay was 800 μM and for studies of NAD/NADH/spermidine effect on binding was 293 μM. In all the above-described assays, a series of 16 2-fold dilutions of the unlabeled protein in MST buffer was prepared, and the labeled DHPS concentration was constant in all samples (100 nM). The measurements were done with NanoTemper Monolith in the red channel set as follows: nanoRED 15% intensity, medium MST power, 25°C, before MST 3 s, 20 s MST on time, 1 s after MST. For every assay data from four measurements was averaged and the signal after 1.5 s was analyzed in MO.Affinity Analysis 3 software (NanoTemper Technologies). For complex stoichiometry evaluation the labeling approach was as described above. The MST buffer was supplemented with 1uM NAD to lower the Kd and generate a more clearly resolved saturation kink. To keep the concentration of the labeled target high enough relative to Kd and not saturate the detector 1 μM of the labeled DHS and 399 μM of unlabeled DHS were used (per monomer concentrations, 100 μM of tetramer in total). A series of 24 dilutions of ERK2 (without 6xHis-tag) was designed to best cover the area surrounding the point of saturation. The measurements were performed with NanoTemper Monolith as described above but the excitation light intensity was set to 2%. Data from four measurements was averaged in MO. Affinity Analysis 3 software and linear regression was performed for linear areas surrounding the saturation kink with Prism (GraphPad). In vitro deoxyhypusination assay The reaction was done in 0.1 M glycine/NaOH pH 9.5 buffer supplemented with 1 mM DTT, 1 mM NAD and 1 mM spermidine at RT. For the time course experiments, 50 nM DHPS and 30 μM eIF5A1 were used and 100 μM of ERK2 or BSA (AppliChem) was added to appropriate samples. The reaction was started by the addition of eIF5A1 with a multichannel pipet. In the same manner, 10 μL samples from all the reactions were taken at appropriate timepoints and immediately mixed with 2 μL o 6x sample buffer for Western blotting. The analysis of the effect of ERK2 concentration was executed in the same way but different concentrations of ERK2 were used and the samples were taken only after 8.5 min. Samples were denatured at 95°C for 5 min, and 6 μL of each sample was separated on 12% SDS-PAGE gel for immunoblotting. Panceau S signal was used for chemiluminescent signal normalization. All experiments were done in triplicates.