Supplementary Figure 4: Single-site mutations of HCA and HCHA adopt wild-type-like structures.
From: N-linked glycosylation of SV2 is required for binding and uptake of botulinum neurotoxin A
The thermal stability of proteins was measured using a fluorescence-based thermal shift assay on a StepOne real-time PCR system (ThermoFisher). Specifically, protein melting was monitored using a hydrophobic dye, SYPRO Orange (Sigma-Aldrich), as the temperature was increased in a linear ramp from 20oC to 95oC. The midpoint of the protein-melting curve (Tm) was determined using the software provided by the instrument manufacturer. The data are presented as mean ± S.D., n = 3. All HCA and HCHA mutants showed Tm values comparable to the wild-type protein, indicating correct protein folding.
