Mulligan Vikram Khipple, Kerman Aaron, Ho Sylvia, Chakrabartty Avijit
Department of Biochemistry, Ontario Cancer Institute, University of Toronto, Toronto Medical Discovery Tower 4-305, MaRS Building, 101 College Street, Toronto, Ontario, Canada M5G 1L7.
J Mol Biol. 2008 Nov 7;383(2):424-36. doi: 10.1016/j.jmb.2008.08.024. Epub 2008 Aug 22.
Mutations in the Cu,Zn superoxide dismutase (SOD1) cause a subset of amyotrophic lateral sclerosis cases. SOD1 is a homodimer in which each monomer binds one copper atom and one zinc atom. Mutation is believed to increase the conformational flexibility of SOD1, giving rise to a misfolded SOD1 population with novel cytotoxic properties. While SOD1's metal ligands affect its stability greatly, little is known about the role these metals play in the folding, unfolding, and misfolding processes. Here, we present a method by which we were able to measure the rates of metal release during SOD1 unfolding in guanidine hydrochloride. Rates of dimer dissociation, measured by a time-resolved cross-linking assay, and conformational changes in SOD1's beta-barrel core, monitored by tryptophan fluorescence intensity, were compared with the rates of copper release and zinc release. Correlations were observed across a range of denaturant concentrations, giving rise to a more detailed model of the SOD1 unfolding process than was previously available. According to this model, the major unfolding pathway involves simultaneous dimer dissociation and zinc release as an early step that is followed by a slow conformational change in the protein's core, which, in turn, is followed by rapid copper release. This model establishes a zinc-deficient, copper-loaded SOD1 monomer as a well-populated SOD1 unfolding intermediate and a species likely to be populated under conditions of denaturational stress. Because the cytotoxicity of zinc-deficient SOD1 has been demonstrated previously, this species is a good candidate for the cytotoxic species in SOD1-associated amyotrophic lateral sclerosis.
铜锌超氧化物歧化酶（SOD1）的突变导致了一部分肌萎缩侧索硬化症病例。SOD1是一种同型二聚体，其中每个单体结合一个铜原子和一个锌原子。据信，突变会增加SOD1的构象灵活性，从而产生具有新细胞毒性特性的错误折叠的SOD1群体。虽然SOD1的金属配体对其稳定性有很大影响，但对于这些金属在折叠、去折叠和错误折叠过程中所起的作用知之甚少。在这里，我们提出了一种方法，通过该方法我们能够测量在盐酸胍中SOD1去折叠过程中金属释放的速率。通过时间分辨交联测定法测量的二聚体解离速率，以及通过色氨酸荧光强度监测的SOD1β桶核心的构象变化，与铜释放速率和锌释放速率进行了比较。在一系列变性剂浓度范围内观察到了相关性，从而产生了一个比以前更详细的SOD1去折叠过程模型。根据这个模型，主要的去折叠途径包括早期同时发生的二聚体解离和锌释放，随后是蛋白质核心的缓慢构象变化，接着是快速的铜释放。这个模型确立了一种锌缺乏、铜负载的SOD1单体作为一种大量存在的SOD1去折叠中间体，以及一种可能在变性应激条件下大量存在的物种。由于先前已经证明了锌缺乏的SOD1具有细胞毒性，因此这个物种是SOD1相关肌萎缩侧索硬化症中细胞毒性物种的一个很好的候选者。
