Monolith nt 115 series instrument

Binding experiments were carried out by microscale thermophoresis with a Monolith NT.115 Series instrument (Nano Temper Technologies). The 6His-PASTA4 domain was labelled using the Monolith Protein Labeling Kit RED-NHS according to the manufacturer’s instructions. Briefly, 16 nM of labelled 6His-PASTA4 mixed (1:1 v/v) with increasing concentrations of either 6His-LytB_NM (from 818 μM to 0.025 μM), 6His-LytB_C (from 930 μM to 0.0284 μM), 6His-LytB_N (from 1210 μM to 0.037 μM) or 6His-LytB_Nmut (from 326 μM to 0.00994 μM μM) were loaded into standard Monolith NT.115 capillaries and MST was measured at RT in buffer 20mM Tris HCl pH8, 0.5M Choline, 1mM DTT, 0.5mM EDTA, 0.1 % Tween 20. Analysis was performed with the Monolith software. The dissociation constant (Kd) to measure affinity was quantified by analysing the change in the fraction bound as a function of the ligand concentration. In order to calculate the fraction bound, all ΔFnorm (normalized fluorescence = fluorescence after thermophoresis/initial fluorescence) values of a curve are divided by the curve amplitude, resulting in the fraction bound (from 0 to 1) for each data point. These experiments were biologically and technically made in triplicates.

Microscale thermophoresis (MST) measurements were performed on a Monolith NT.115 series instrument (NanoTemper). Prior to thermophoresis measurements proteins were labeled by incubation with Cy5-NHS (Lumiprobe) for 30 min at room temperature. Unreacted dye was separated from the protein using PD SpinTrap™ G-25 gel filtration columns (GE Healthcare) according to the manufacturer’s protocol. Serial dilutions of the cap analogues (starting from 200 μM of cap analog) in MST reaction buffer (20 mM HEPES, 50 mMKCl, 0.2 mM EDTA, 0.01% Triton-X, 700 μM mercaptoethanol, 0.01% Tween-20, pH = 8) were prepared and mixed with an equal volume of the labeled protein (^~50 nM). The mixture was filled into premium coated capillaries (4 μL) and directly measured. MST power was set to 30–40%, LED power was set to 20% Red (Exc.: 625nm, Em.: 680 nm). Thermophoresis measurements were performed with the following settings: fluorescence before (5 s), MST on (30 s), fluorescence after (3 s). The capillaries were measured three times in direct succession as technical replicates. MST data were normalised to baseline differences and K_d values were calculated using nonlinear regression assuming a Hill coefficient of 1.0 (GraphPad Prism). MST is known to produce occasional outliers. This was handled as follows: 16 data points were measured per binding curve and at least 12 data point were used for each fit.