Multimode nanoscope 5

Samples were diluted 1:1,000 with ultrapure water (Milli-Q^®, MA, United States) and dropped onto freshly cleaved mica plates, with the addition of 10 µl of 10 mM CaCl₂ solution. After 15 min, plates were washed with ultrapure water, and the excess was removed with filter paper (Whatman, Germany). Images were obtained at room temperature by a Multimode-Nanoscope V (Veeco, Santa Barbara, CA, United States) from the Nanoscopy Laboratory from the Theoretical and Applied Physical Chemistry Research Institute (INIFTA, CONICET, La Plata, Argentina). The microscope was operated in tapping mode, with an etched silicon probe model RTesp-Bruker (cantilever resonance frequency: 300 kHz; force constant 42 N/m; tip radius 8–12 nm). Typical scan rates were carried out at 1 Hz.

Wt and Arg173Pro (0.2 mg/mL) proteins were incubated with 0.2 mM SDS (either at pH 7.4 or 5.0) for 48 h at 37°C. Complex formation was detected by the binding to ThT as described above. Protein binding of heparin was tested following incubation of 0.2 mg/mL proteins with heparin at a 2:1 heparin/protein molar ratio for 48 h at 37°C. In order to confirm whether binding to heparin results in the formation of high molecular weight complexes, the incubation mixtures were analyzed by PAGGE. In a separate experiment, the Arg173Pro variant was incubated at 0.5 mg/mL and pH 7.4 for 24 h at 37°C in the absence or in the presence of heparin, and morphology was compared by atomic force microscopy (AFM) as described previously [13 (link)]. All images were obtained at room temperature using a Multimode-Nanoscope V (Veeco, Santa Barbara, CA) operating in tapping mode with an etched silicon Probe model Arrow-NCR-50 Nano World (cantilever resonance frequency: 258 kHz, Force constant 42 N/m; tip radius 5–10 nm). Typical scan rates were 1–1.5 Hz.

AFM measurements were performed on MultiMode Nanoscope V (Veeco, Plainview, NY, USA) operating in tapping mode. Noncontact polysilicon cantilevers with high aspect ratio were used (TipsNano, Estonia, resonant frequency—120 kHz, Q-factor—350). Image processing was performed using Gwyddion (Chechen) software (Version 2.62).