Nanostar

Nup98^FG samples were prepared at a concentration of 2.5 μM in 50 mM sodium phosphate buffer containing 1 mM TCEP at pH 3. Samples at pH 7 were prepared in the same buffer by adjusting the pH to 7 just before measurement and reducing the protein concentration to 100 nM. Both concentrations were optimized to avoid saturation of the detector. Measurements were conducted at 25 °C using a DynaPro NanoStar instrument (Wyatt Technologies) and NanoStar disposable microcuvettes. The samples were illuminated with a 120 mW air-launched laser at a wavelength of 662 nm and the intensity of light scattered at an angle of 90° was detected with an actively quenched, solid-state single-photon counting module. Data were acquired with an acquisition time of 5 s with a total of five acquisitions per measurement. The hydrodynamic radii were determined using the Dynamics (version 7.10.0.23) software package. The final values are given as the average and standard error of 12 measurements.

Autocorrelation data were collected in a NanoStar instrument (Wyatt Technology, Santa Barbara, CA). One hundred–microliter samples were inserted into a 1-μl quartz cuvette (WNQC01-00, Wyatt Instruments), using excess sample to minimize the impact of evaporation in the observation chamber. Laser light scattering was measured at 658 nm at a detection angle of 90°. For the temperature scans, a ramp rate of 1°/min was applied with 5-s data acquisitions and averaging three replicates for each temperature point. Data were collected and processed by using Dynamics 7.4 (Wyatt Technology) and SEDFIT.

Dynamic light scattering experiments were performed with a DynaPro^® NanoStar^® instrument (WYATT technology, Santa Barbara, CA, USA). Data analysis was performed with the DYNAMICS software package. The analyzed correlation curves were averages of ten measurements of 7 s.