Kayatekin Can, Zitzewitz Jill A, Matthews C Robert
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
J Mol Biol. 2008 Dec 12;384(2):540-55. doi: 10.1016/j.jmb.2008.09.045. Epub 2008 Sep 26.
Over 100 amino acid replacements in human Cu,Zn superoxide dismutase (SOD) are known to cause amyotrophic lateral sclerosis, a gain-of-function neurodegenerative disease that destroys motor neurons. Supposing that aggregates of partially folded states are primarily responsible for toxicity, we determined the role of the structurally important zinc ion in defining the folding free energy surface of dimeric SOD by comparing the thermodynamic and kinetic folding properties of the zinc-free and zinc-bound forms of the protein. The presence of zinc was found to decrease the free energies of a peptide model of the unfolded monomer, a stable variant of the folded monomeric intermediate, and the folded dimeric species. The unfolded state binds zinc weakly with a micromolar dissociation constant, and the folded monomeric intermediate and the native dimeric form both bind zinc tightly, with subnanomolar dissociation constants. Coupled with the strong driving force for the subunit association reaction, the shift in the populations toward more well-folded states in the presence of zinc decreases the steady-state populations of higher-energy states in SOD under expected in vivo zinc concentrations (approximately nanomolar). The significant decrease in the population of partially folded states is expected to diminish their potential for aggregation and account for the known protective effect of zinc. The approximately 100-fold increase in the rate of folding of SOD in the presence of micromolar concentrations of zinc demonstrates a significant role for a preorganized zinc-binding loop in the transition-state ensemble for the rate-limiting monomer folding reaction in this beta-barrel protein.
已知人类铜锌超氧化物歧化酶（SOD）中有100多种氨基酸置换会导致肌萎缩侧索硬化症，这是一种功能获得性神经退行性疾病，会破坏运动神经元。假设部分折叠状态的聚集体是毒性的主要原因，我们通过比较无锌和锌结合形式蛋白质的热力学和动力学折叠特性，确定了结构上重要的锌离子在定义二聚体SOD折叠自由能表面中的作用。发现锌的存在会降低未折叠单体的肽模型、折叠单体中间体的稳定变体以及折叠二聚体物种的自由能。未折叠状态以微摩尔解离常数与锌弱结合，折叠单体中间体和天然二聚体形式都与锌紧密结合，解离常数低于纳摩尔。加上亚基缔合反应的强大驱动力，在锌存在下向更良好折叠状态的群体转移会降低在预期体内锌浓度（约纳摩尔）下SOD中高能状态的稳态群体。部分折叠状态群体的显著减少预计会降低它们聚集的可能性，并解释锌已知的保护作用。在微摩尔浓度的锌存在下，SOD折叠速率增加约100倍，这表明在这个β桶蛋白中，一个预先组织好的锌结合环在限速单体折叠反应的过渡态系综中起重要作用。
