Kristensen Line G, Holton James M, Rad Behzad, Chen Yan, Petzold Christopher J, Gupta Sayan, Ralston Corie Y
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA.
Molecular Foundry Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA.
J Synchrotron Radiat. 2021 Sep 1;28(Pt 5):1333-1342. doi: 10.1107/S1600577521004744. Epub 2021 Jul 20.
In the method of X-ray footprinting mass spectrometry (XFMS), proteins at micromolar concentration in solution are irradiated with a broadband X-ray source, and the resulting hydroxyl radical modifications are characterized using liquid chromatography mass spectrometry to determine sites of solvent accessibility. These data are used to infer structural changes in proteins upon interaction with other proteins, folding, or ligand binding. XFMS is typically performed under aerobic conditions; dissolved molecular oxygen in solution is necessary in many, if not all, the hydroxyl radical modifications that are generally reported. In this study we investigated the result of X-ray induced modifications to three different proteins under aerobic versus low oxygen conditions, and correlated the extent of damage with dose calculations. We observed a concentration-dependent protecting effect at higher protein concentration for a given X-ray dose. For the typical doses used in XFMS experiments there was minimal X-ray induced aggregation and fragmentation, but for higher doses we observed formation of covalent higher molecular weight oligomers, as well as fragmentation, which was affected by the amount of dissolved oxygen in solution. The higher molecular weight products in the form of dimers, trimers, and tetramers were present in all sample preparations, and, upon X-ray irradiation, these oligomers became non-reducible as seen in SDS-PAGE. The results provide an important contribution to the large body of X-ray radiation damage literature in structural biology research, and will specifically help inform the future planning of XFMS, and well as X-ray crystallography and small-angle X-ray scattering experiments.
在X射线足迹质谱法（XFMS）中，用宽带X射线源照射溶液中微摩尔浓度的蛋白质，然后使用液相色谱质谱法对产生的羟基自由基修饰进行表征，以确定溶剂可及位点。这些数据用于推断蛋白质在与其他蛋白质相互作用、折叠或配体结合时的结构变化。XFMS通常在有氧条件下进行；溶液中溶解的分子氧对于许多（如果不是所有）通常报道的羟基自由基修饰是必需的。在本研究中，我们研究了在有氧和低氧条件下X射线对三种不同蛋白质诱导修饰的结果，并将损伤程度与剂量计算相关联。对于给定的X射线剂量，我们在较高蛋白质浓度下观察到浓度依赖性保护作用。在XFMS实验中使用的典型剂量下，X射线诱导的聚集和碎片化最小，但对于更高剂量，我们观察到形成了共价高分子量寡聚物以及碎片化，这受到溶液中溶解氧量的影响。二聚体、三聚体和四聚体形式的较高分子量产物存在于所有样品制剂中，并且在X射线照射后，这些寡聚物在SDS-PAGE中显示为不可还原。这些结果为结构生物学研究中的大量X射线辐射损伤文献做出了重要贡献，并且将特别有助于为XFMS以及X射线晶体学和小角X射线散射实验的未来规划提供信息。
