Museth Anna Katrine, Brorsson Ann-Christin, Lundqvist Martin, Tibell Lena A E, Jonsson Bengt-Harald
Division of Molecular Biotechnology, Department of Physics, Chemistry and Biology, Linköping University, SE-581 83 Linköping, Sweden.
Biochemistry. 2009 Sep 22;48(37):8817-29. doi: 10.1021/bi900703v.
More than 100 distinct mutations in the gene (SOD1) for human copper-zinc superoxide dismutase (CuZnSOD) have been associated with familial amyotrophic lateral sclerosis (fALS). Studies of these mutant proteins, which often have been performed under far from physiological conditions, have indicated effects on protein stabilities, catalytic activity, and metal binding affinities but with no common pattern. Also, with the knowledge that ALS is a late onset disease it is apparent that protein interactions which contribute to the disorder might, in the natural cellular milieu, depend on a delicate balance between intrinsic protein properties. In this study, we have used experimental conditions as near as possible to the in vivo conditions to reduce artifacts emanating from the experimental setup. Using 1H-15N HSQC NMR spectroscopy, we have analyzed hydrogen exchange at the amide groups of wild-type (wt) CuZnSOD and the fALS-associated G93A SOD variant in their fully metalated states. From analyses of the exchange pattern, we have characterized the local dynamics at 64% of all positions in detail in both the wt and G93A protein. The results show that the G93A mutation had no effect on the dynamics at a majority of the investigated positions. However, the mutation results in local destabilization at the site of the mutation and also in stabilization at a few positions that were apparently scattered over the entire protein surface. Most remarkably, the mutation selectively destabilized the remote metal binding region. The results indicate that the metal binding region may affect the intermolecular protein-protein interactions which cause formation of protein aggregates.
人类铜锌超氧化物歧化酶（CuZnSOD）基因（SOD1）中100多种不同的突变与家族性肌萎缩侧索硬化症（fALS）相关。对这些突变蛋白的研究，通常是在远非生理条件下进行的，这些研究表明了它们对蛋白质稳定性、催化活性和金属结合亲和力的影响，但没有共同模式。此外，鉴于肌萎缩侧索硬化症是一种迟发性疾病，显然在天然细胞环境中，导致该疾病的蛋白质相互作用可能取决于内在蛋白质特性之间的微妙平衡。在本研究中，我们使用了尽可能接近体内条件的实验条件，以减少实验设置产生的假象。利用1H-15N HSQC核磁共振波谱，我们分析了野生型（wt）CuZnSOD和与fALS相关的G93A SOD变体在其完全金属化状态下酰胺基团的氢交换。通过对交换模式的分析，我们详细表征了wt和G93A蛋白中所有位置64%处的局部动力学。结果表明，G93A突变对大多数研究位置的动力学没有影响。然而，该突变导致突变位点局部不稳定，同时也导致一些明显分散在整个蛋白质表面的位置稳定。最显著的是，该突变选择性地使远程金属结合区域不稳定。结果表明，金属结合区域可能影响导致蛋白质聚集体形成的分子间蛋白质-蛋白质相互作用。
