Nanodsf capillaries

Proteins mixed to a final concentration of 30 µM in presence of 200 µM PIP₂ and incubated overnight at 4 °C were used to fill two standard grade NanoDSF capillaries (Nanotemper) and loaded into a Prometheus NT.48 device (Nanotemper) controlled by PR.ThermControl (version 2.1.2). Excitation power was pre-adjusted to get fluorescence readings between 2000 and 20000 RFU for fluorescence at 330 and 350 nm (F330 and F350, respectively), and samples heated from 20 °C to 90 °C with a slope of 1 °C/min. An apparent Tm was calculated from the inflection points of the fluorescence curves (Ratio F350/F330) for ANTH and ENTH monomeric samples, where ΔT_m = T_m mutant − T_m wild-type. To ensure we did not see any oligomerization interference effect potentially caused by domain instability, we discarded all mutants with a ΔT_m larger than 2 °C in our complex assembly study.
The scattering signal recorded (backscattering mode) was used as a stability reporter for the AENTH samples, where the mid aggregation point, T_agg, corresponds to the inflexion points in the scattering curves of the first transitions observed upon heating (T_agg = mid-aggregation temperature obtained from scattering curves). ΔT_agg was calculated in the same way as done for ΔT_m: ΔT_agg = T_agg mutant – T_agg wild-type.

Purified Arr proteins were diluted with SEC buffer to a concentration of 30 µM for analysis by NanoDSF. The compounds RMP, RBT, AMK, and 5j were added to a final concentration of 300 µM, equivalent to a final concentration of 1% DMSO, which was included as a control. Samples were loaded into standard grade NanoDSF capillaries (Nanotemper) and loaded into a Prometheus NT.48 device (Nanotemper) controlled by PR.ThermControl (version 2.1.2). An excitation power of 50% was used to obtain fluorescence readings above 2,000 RFU for F330 and F350. Samples were heated from 20 to 90°C with a slope of 1°C/min. Melting experiments were done in triplicate.