Rumfeldt Jessica A O, Stathopulos Peter B, Chakrabarrty Avijit, Lepock James R, Meiering Elizabeth M
Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry and Department of Chemistry, University of Waterloo, Waterloo, Ont. Canada N2L 3G1.
J Mol Biol. 2006 Jan 6;355(1):106-23. doi: 10.1016/j.jmb.2005.10.042. Epub 2005 Nov 8.
Mutations in human copper zinc superoxide dismutase (hSOD) that are associated with amyotrophic lateral sclerosis (ALS) have been proposed to destabilize the protein and thereby enhance toxic protein aggregation. In previous studies, denaturation of metallated (holo) hSODs was found to be irreversible, and complicated by the formation of intermolecular disulfide bonds. Here, ALS-associated mutations (E100G, G93A, G85R and A4V) are introduced into a pseudo wild-type background containing no free cysteine residues. The guanidinium chloride-induced denaturation of the holo proteins is generally found to be highly reversible (except for A4V, which tended to aggregate), enabling quantitative analysis of the effects of the mutations on protein stability. Denaturation and renaturation curves were monitored by tryptophan fluorescence, circular dichroism, enzyme activity, chemical cross-linking and analytical sedimentation, as a function of equilibration time and protein concentration. There is strong kinetic hysteresis, with curves requiring exceptionally long times (many days for pseudo wild-type) to reach equilibrium, and evidence for the formation of kinetic and equilibrium intermediate(s), which are more highly populated at lower protein concentrations. The effects of metal dissociation were included in the data fitting. The full protein concentration dependence is best described using a three-state model involving metallated native dimer, metallated monomeric intermediate and unfolded monomers with no bound metals; however, at high protein concentrations the unfolding approaches a two-state transition with metal binding to both the native dimers and unfolded monomers. We show that the E100G, G93A and G85R mutations decrease overall protein stability, largely by decreasing monomer stability with little effect on dimer dissociation. Comparison of the chemical denaturation data with ALS disease characteristics suggests that aggregation of some mutant hSOD may occur through increased population of partially folded states that are less stable than the monomeric intermediate and accessed from the destabilized holo protein.
与肌萎缩侧索硬化症（ALS）相关的人类铜锌超氧化物歧化酶（hSOD）突变被认为会使蛋白质不稳定，从而增强有毒蛋白质聚集。在先前的研究中，发现金属化（全酶）hSOD的变性是不可逆的，并且因分子间二硫键的形成而变得复杂。在此，将ALS相关突变（E100G、G93A、G85R和A4V）引入不含游离半胱氨酸残基的假野生型背景中。一般发现，氯化胍诱导的全酶蛋白变性是高度可逆的（除了倾向于聚集的A4V），这使得能够对突变对蛋白质稳定性的影响进行定量分析。通过色氨酸荧光、圆二色性、酶活性、化学交联和分析沉降监测变性和复性曲线，作为平衡时间和蛋白质浓度的函数。存在强烈的动力学滞后现象，曲线需要极长的时间（假野生型需要很多天）才能达到平衡，并且有证据表明形成了动力学和平衡中间体，在较低蛋白质浓度下这些中间体的数量更多。数据拟合中包括了金属解离的影响。使用涉及金属化天然二聚体、金属化单体中间体和无结合金属的未折叠单体的三态模型可以最好地描述整个蛋白质浓度依赖性；然而，在高蛋白质浓度下，展开接近二态转变，金属与天然二聚体和未折叠单体都结合。我们表明，E100G、G93A和G85R突变降低了整体蛋白质稳定性，主要是通过降低单体稳定性，而对二聚体解离影响很小。将化学变性数据与ALS疾病特征进行比较表明，一些突变型hSOD的聚集可能是通过部分折叠状态的数量增加而发生的，这些部分折叠状态比单体中间体不稳定，并且是从不稳定的全酶蛋白中产生的。
