Lysozyme conformational changes with ionic liquids – FTIR, fluorescence, SAXS and crystallography study

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.

小角X射线散射（Small-Angle X-ray Scattering，SAXS）实验于澳大利亚墨尔本的澳大利亚同步辐射装置SAXS/WAXS光束线开展。该实验装置搭载自动化多孔板进样系统，可将每份待测样品与空白溶剂依次吸入同一毛细管内，从而实现高精度的缓冲液背景扣除。每份处于流动状态的样品均采集10幅连续的1秒曝光帧。这套系统专为弱散射蛋白质样品设计，通过降低每个分子所受的X射线辐射剂量，有效减少了蛋白质的辐射损伤。溶菌酶样品在测量前需平衡1小时，每份样品取50 μL载入自动化进样所用的96孔板中。所有SAXS实验的q范围为0.006至0.53 Å⁻¹。数据处理采用Scatterbrain 2.82软件，SAXS数据分析则依托ATSAS软件包中的Chromixs、DAMMIN及SREFLEX工具完成。SAXS散射曲线以经精准溶剂背景扣除后的多次测量平均值形式呈现。通过ATSAS软件包的吉尼耶（Guinier）近似法，计算得到溶菌酶的回转半径（radius of gyration，Rg）。此外，还利用ATSAS软件获取了距离分布函数P(r)与最大直径Dmax。采用CRYSOL软件对不同溶菌酶晶体结构（蛋白质数据库（Protein Data Bank，PDB）编号：1dpw、1dpx、1lkr、1lks、1uco、3a8z、3ru5、3wul、3wum、3wun及193l）进行匹配筛选，最终选取与SAXS散射曲线匹配度最高的1dpw结构。文中给出了初始结构与SAXS散射曲线的卡方（χ²）偏差值，并基于χ²值筛选出不同离子液体环境下的最优精修模型。