Lysozyme conformational changes with ionic liquids – FTIR, fluorescence, SAXS and crystallography study
datasetposted on 03.12.2020, 00:33 by Hank HanHank Han, Kate Smith, Connie Darmanin, Timothy M. Ryan, Tamar GreavesTamar Greaves, Calum Drummond
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. Lysozyme 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 lysozyme was 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 lysozyme crystal structures (PDB ID 1dpw, 1dpx, 1lkr, 1lks, 1uco, 3a8z, 3ru5, 3wul, 3wum, 3wun and 193l), and 1dpw was selected as the closest match to the SAXS patterns. The discrepancies χ2 values of the initial structure with the SAXS patterns were provided, while the best refined models in different ILs were selected based on the χ2 values.
Read the peer-reviewed publication
Qi Han, Kate M. Smith, Connie Darmanin, Timothy M. Ryan, Calum J. Drummond, Tamar L. Greaves, Lysozyme conformational changes with ionic liquids: Spectroscopic, small angle x-ray scattering and crystallographic study, Journal of Colloid and Interface Science, Volume 585, 2021, Pages 433-443, ISSN 0021-9797