Standard treated capillaries

MST experiments were carried out in a Monolith NT.115 device using standard treated capillaries (NanoTemper Technologies). Fluorescence changes resulting from thermophoresis were recorded using blue channel optics of the instrument (λ_ex = 470 ± 15 nm, λ_em = 520 ± 10 nm) for the 30 s period of infrared laser heating at 40% of maximum laser power followed by a 5 s of cooling period. Measurements were performed in a buffer containing 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM MgCl₂, 1 mM DTT and 0.1% Tween 20. 2'-O-(6-[Fluoresceinyl]aminohexylcarbamoyl)-cyclic diguanosine monophosphate (2’fluo-AHC-c-di-GMP, Biolog, Bremen, Germany) was added to probe the binding affinity of CleD domains. A 16-point 1:1 serial dilution series of proteins was titrated against a fixed concentration (60 nM) of 2’fluo-AHC-c-di-GMP. Data were evaluated in the program ProFit (Quansoft, Zurich, Switzerland) and fitted using the Hill equation.

Binding affinities between FH19-20 or FH (from Complement Technologies, US) and wild type and mutants of FhbA proteins was determined using Microscale Thermophoresis (MST) with a Monolith NT.115 instrument (Nanotemper Technologies, Germany). FH19-20 and FH were labelled with RED-tris-NTA dye (Nanotemper Technologies, US) in PBS according to the manufacturer’s instructions. 10 μl of 300 nM labelled protein was mixed with 10 μl of ligand in PBS/0.025% Tween-20, the mixture loaded into standard treated capillaries (Nanotemper Technologies, US) and thermophoresis was measured at 22° C for 22–30 s with 20% LED power and 20%/60% infrared laser power. Three independent measurements were made, and results were analysed using the MO. Affinity Analysis software version 2.1 (Nanotemper Technologies, US). For gel filtration 100 μl of proteins (20 nmole) were eluted individually and in combination with FH19-20 on a Superdex 200 Increase 10/300 GL column attached to an ÄKTA (GE-healthcare) with PBS buffer at +4° C. 1 ml fractions from each run were collected and subjected to SDS-PAGE analyses on TGX gradient (4–20%) precast mini-gels (Biorad, CA, US), which were fixed, stained with QC Colloidal Coomassie Stain (Biorad), and proteins were visualized using Image Lab (Biorad). For high salt experiments, PBS buffer supplemented with 500 mM NaCl was used.

The interaction between c-di-GMP nucleotides and various proteins was measured by using MST with a fluorescein-labelled c-di-GMP (2′-Fluo-AHC-c-di-GMP, abbreviated as fl-c-di-GMP; 2′-Fluo-AHC-c-di-AMP, fl-c-di-AMP; 2′-Fluo-AHC-cGMP, fl-cGMP; Biolog, Germany). These chemicals contain carboxyfluorescein that has excitation and emission wavelengths of 497 nm and 520 nm, respectively, which can be detected directly by a MST instrument. The experiments were performed on a Monolith NT.115 device using standard treated capillaries (NanoTemper Technologies, Germany). The concentration of the particular protein varied from 0.036 to 150 μM with a 2-fold gradient and the concentration of fl-c-di-GMP was constant at 20 nM. Fluorescence intensity due to thermophoresis was recorded using the blue channel optics of the instrument (λ_ex = 470 ± 15 nm, λ_em = 520 ± 10 nm) during a 30 s period of infrared laser heating at 80% of the maximum laser power, followed by a 5 s cooling period. Measurements were performed in buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM MgCl₂, 2 mM DTT and 0.05% Tween 20. The KD Fit of NanoTemper Analysis software (ver. 1.5.41) was used for fitting the curve and calculation of the dissociation constant (K_d). Assays were repeated with at least three biological replicates in triplicate.

The purified TLR4 ectodomain and soluble CD14 were labelled with DyLight-488. The labelling reaction was performed according to the manufacturer’s instructions. Full-length TLR4, CD14 and MD2 were obtained from HEK293 lysates. The GFP-tagged TLR4, CD14 and MD2 constructs were transfected into HEK293, incubated for 48 h and lysed in RIPA buffer and clear supernatant was obtained by the centrifugation.
The labelled TLR4 or CD14 was adjusted to 20 nM with MST buffer, and the lysates were diluted according to their fluorescence intensity. Recombinant CnB was dissolved in MST buffer to the appropriate concentration. A series of 16 1:1 dilutions were prepared, and the labelled proteins or GFP-tagged receptors from the lysates were added to each ligand dilution and mixed. After 10-min incubation, each solution was added to Standard Treated Capillaries (NanoTemper Technologies). Thermophoresis was measured using a Monolith NT.115 instrument (NanoTemper Technologies) at an ambient temperature of 25 °C with 5 s/30 s/5 s laser off/on/off times, respectively. The instrument parameters were adjusted to 50% LED power and 20% MST power. The data from three independently pipetted measurements were analysed (NT.Analysis software version 1.5.41, NanoTemper Technologies) using the signal from Thermophoresis + T-Jump36 (link)37 (link).

MicroScale Thermophoresis (MST) was performed using purified CqsR-LBD. For MST experiments, nickel affinity resin-purified CqsR-LBD was further purified using anion exchange chromatography (Source15Q—GE Healthcare) and size exclusion chromatography (S200 –GE Healthcare). Purified CqsR-LBD was concentrated to 4.3 mg/ml and stored in 50 mM sodium phosphate, pH 7.7, and 300 mM sodium chloride. CqsR-LBD was diluted to 20 μM and labeled using the Monolith NT Protein Labeling Ki RED-NHS (NanoTemper Technologies). Different concentrations of ethanolamine were incubated with 20 nM working stock solutions of labeled protein in the dark for 30 min at 4° C. After incubation, the samples were transferred into standard treated capillaries (NanoTemper Technologies) and read in a Monolith NT.115 Blue/Red instrument at room temperature using 20% LED and medium MST power. Binding affinities were calculated from three experiments.