Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, USA.
The Pasarow Mass Spectrometry Laboratory, University of California, Los Angeles, CA, USA.
J Am Soc Mass Spectrom. 2019 Feb;30(2):218-226. doi: 10.1007/s13361-018-2075-y. Epub 2018 Oct 16.
Solvent-accessibility change plays a critical role in protein misfolding and aggregation, the culprit for several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Mass spectrometry-based hydroxyl radical (·OH) protein footprinting has evolved as a powerful and fast tool in elucidating protein solvent accessibility. In this work, we used fast photochemical oxidation of protein (FPOP) hydroxyl radical (·OH) footprinting to investigate solvent accessibility in human copper-zinc superoxide dismutase (SOD1), misfolded or aggregated forms of which underlie a portion of ALS cases. ·OH-mediated modifications to 56 residues were detected with locations largely as predicted based on X-ray crystallography data, while the interior of SOD1 β-barrel is hydrophobic and solvent-inaccessible and thus protected from modification. There were, however, two notable exceptions-two closely located residues inside the β-barrel, predicted to have minimal or no solvent accessibility, that were found modified by FPOP (Phe20 and Ile112). Molecular dynamics (MD) simulations were consistent with differential access of peroxide versus quencher to SOD1's interior complicating surface accessibility considerations. Modification of these two residues could potentially be explained either by local motions of the β-barrel that increased peroxide/solvent accessibility to the interior or by oxidative events within the interior that might include long-distance radical transfer to buried sites. Overall, comparison of modification patterns for the metal-free apoprotein versus zinc-bound forms demonstrated that binding of zinc protected the electrostatic loop and organized the copper-binding site. Our study highlights SOD1 hydrophobic groups that may contribute to early events in aggregation and discusses caveats to surface accessibility conclusions. Graphical Abstract.
溶剂可及性变化在蛋白质错误折叠和聚集中起着关键作用，这是包括肌萎缩侧索硬化症（ALS）在内的几种神经退行性疾病的罪魁祸首。基于质谱的羟基自由基（·OH）蛋白质足迹法已成为阐明蛋白质溶剂可及性的强大而快速的工具。在这项工作中，我们使用快速光化学氧化蛋白质（FPOP）羟基自由基（·OH）足迹法来研究人类铜锌超氧化物歧化酶（SOD1）的溶剂可及性，其错误折叠或聚集形式是部分 ALS 病例的基础。使用·OH 介导的修饰检测到 56 个残基，其位置主要基于 X 射线晶体学数据进行预测，而 SOD1β-桶的内部是疏水性的，溶剂不可及的，因此免受修饰。然而，有两个值得注意的例外——β-桶内两个位置非常接近的残基，预计溶剂可及性最小或没有，通过 FPOP 发现被修饰（Phe20 和 Ile112）。分子动力学（MD）模拟与过氧化物和淬灭剂对 SOD1 内部的不同可及性一致，这使得表面可及性考虑变得复杂。这两个残基的修饰可能有两种解释，要么是β-桶的局部运动增加了过氧化物/溶剂对内部的可及性，要么是内部的氧化事件，可能包括长距离自由基转移到埋藏部位。总体而言，比较金属游离蛋白与锌结合形式的修饰模式表明，锌的结合保护了静电环并组织了铜结合位点。我们的研究强调了 SOD1 疏水区，它们可能导致聚集的早期事件，并讨论了表面可及性结论的注意事项。图表摘要。
