Hauptman-Woodward Medical Research Institute, Buffalo, New York, United States of America.
Department of Cell Stress Biology, Roswell Park Comprehensive Cancer Center, Buffalo, New York, United States of America.
PLoS One. 2020 Nov 17;15(11):e0239702. doi: 10.1371/journal.pone.0239702. eCollection 2020.
A significant problem in biological X-ray crystallography is the radiation chemistry caused by the incident X-ray beam. This produces both global and site-specific damage. Site specific damage can misdirect the biological interpretation of the structural models produced. Cryo-cooling crystals has been successful in mitigating damage but not eliminating it altogether; however, cryo-cooling can be difficult in some cases and has also been shown to limit functionally relevant protein conformations. The doses used for X-ray crystallography are typically in the kilo-gray to mega-gray range. While disulfide bonds are among the most significantly affected species in proteins in the crystalline state at both cryogenic and higher temperatures, there is limited information on their response to low X-ray doses in solution, the details of which might inform biomedical applications of X-rays. In this work we engineered a protein that dimerizes through a susceptible disulfide bond to relate the radiation damage processes seen in cryo-cooled crystals to those closer to physiologic conditions. This approach enables a low-resolution technique, small angle X-ray scattering (SAXS), to detect and monitor a residue specific process. A dose dependent fragmentation of the engineered protein was seen that can be explained by a dimer to monomer transition through disulfide bond cleavage. This supports the crystallographically derived mechanism and demonstrates that results obtained crystallographically can be usefully extrapolated to physiologic conditions. Fragmentation was influenced by pH and the conformation of the dimer, providing information on mechanism and pointing to future routes for investigation and potential mitigation. The novel engineered protein approach to generate a large-scale change through a site-specific interaction represents a promising tool for advancing radiation damage studies under solution conditions.
在生物 X 射线晶体学中，一个重大问题是入射 X 射线束引起的辐射化学。这会产生全局和局部损伤。局部损伤会导致对结构模型的生物解释产生偏差。晶体的冷冻冷却已成功减轻了损伤，但不能完全消除损伤；然而，在某些情况下，冷冻冷却可能很困难，并且已经表明它限制了与功能相关的蛋白质构象。X 射线晶体学中使用的剂量通常在千戈瑞到兆戈瑞范围内。虽然二硫键是晶体状态下蛋白质中受影响最严重的物种之一，无论是在低温还是高温下，但关于它们对溶液中低 X 射线剂量的反应的信息有限，这些细节可能会为 X 射线的生物医学应用提供信息。在这项工作中，我们设计了一种通过易受影响的二硫键二聚化的蛋白质，将在冷冻冷却晶体中观察到的辐射损伤过程与更接近生理条件的过程联系起来。这种方法使低分辨率技术，小角 X 射线散射（SAXS）能够检测和监测残基特异性过程。观察到工程蛋白的剂量依赖性片段化，这可以通过二硫键断裂的二聚体到单体的转变来解释。这支持了晶体学衍生的机制，并表明从晶体学获得的结果可以有用地外推到生理条件。片段化受 pH 和二聚体构象的影响，提供了关于机制的信息，并指向未来的研究和潜在缓解途径。通过特定于位置的相互作用产生大规模变化的新型工程蛋白方法代表了在溶液条件下推进辐射损伤研究的有前途的工具。
