Effect of ionic liquids on the fluorescence properties and aggregation of Superfolder Green Fluorescence Protein

SAXS experiments were carried out at the SAXS/WAXS beamline at the Australian Synchrotron, Melbourne, Australia. The setup had an automated well plate system, where each sample and blank solvent was drawn into the same capillary to enable accurate buffer subtraction. Ten successive frames of 1 s exposure were collected for each sample under flow. This system has been designed for weakly-scattering protein samples and reduces protein damage through minimizing X-ray radiation dose per molecule. Superfolder Green Fluorescence Protein (sfGFP) samples were equilibrated for 1 hour before measurement, and 50 μL of each sample were loaded into the 96-well plate used for the automated sampling setup. The q-range for all the SAXS experiments was 0.006 to 0.53 Å−1. Scatterbrain 2.82 was used for SAXS data processing, and Chromixs, DAMMIN and SREFLEX of the ATSAS software package were used for SAXS data analysis. SAXS patterns are presented as the average of the measurements after careful blank subtraction of the corresponding solvent. The radius of gyration (Rg) for sfGFPwas calculated from the Guinier approximation through the ATSAS package. The distance distribution function P(r) and the maximum diameter (Dmax) were also obtained using the ATSAS software. CRYSOL was used to screen different sfGFP crystal structures (PDB ID 2b3p), and 2b3p was selected as the closest match to the SAXS patterns.