Department of Biochemistry and Biophysics, Arrhenius Laboratories of Natural Sciences , Stockholm University , S-106 91 Stockholm , Sweden.
Division of Biochemistry & Structural Biology, Department of Chemistry , Lund University , Box 124, 22100 Lund , Sweden.
J Am Chem Soc. 2018 Dec 5;140(48):16570-16579. doi: 10.1021/jacs.8b08141. Epub 2018 Nov 27.
A conspicuous feature of the amyotrophic lateral sclerosis (ALS)-associated protein SOD1 is that its maturation into a functional enzyme relies on local folding of two disordered loops into a catalytic subdomain. To drive the disorder-to-order transition, the protein employs a single Zn ion. The question is then if the entropic penalty of maintaining such disordered loops in the immature apoSOD1 monomer is large enough to explain its unusually low stability, slow folding, and pathological aggregation in ALS. To find out, we determined the effects of systematically altering the SOD1-loop lengths by protein redesign. The results show that the loops destabilize the apoSOD1 monomer by ∼3 kcal/mol, rendering the protein marginally stable and accounting for its aggregation behavior. Yet the effect on the global folding kinetics remains much smaller with a transition-state destabilization of <1 kcal/mol. Notably, this 1/3 transition-state to folded-state stability ratio provides a clear-cut example of the enigmatic disagreement between the Leffler α value from loop-length alterations (typically 1/3) and the "standard" reaction coordinates based on solvent perturbations (typically >2/3). Reconciling the issue, we demonstrate that the disagreement disappears when accounting for the progressive loop shortening that occurs along the folding pathway. The approach assumes a consistent Flory loop entropy scaling factor of c = 1.48 for both equilibrium and kinetic data and has the added benefit of verifying the tertiary interactions of the folding nucleus as determined by phi-value analysis. Thus, SOD1 not only represents a case where evolution of key catalytic function has come with the drawback of a destabilized apo state but also stands out as a well-suited model system for exploring the physicochemical details of protein self-organization.
肌萎缩侧索硬化症 (ALS) 相关蛋白 SOD1 的一个显著特征是，其成熟为功能性酶依赖于两个无规卷曲环在催化亚结构域中的局部折叠。为了驱动无序到有序的转变，该蛋白使用单个 Zn 离子。那么问题是，如果在不成熟的 apoSOD1 单体中维持这种无规卷曲环的熵罚足够大，是否可以解释其异常低的稳定性、缓慢的折叠和在 ALS 中的病理性聚集。为了找出答案，我们通过蛋白质重新设计确定了系统改变 SOD1 环长度对其的影响。结果表明，这些环使 apoSOD1 单体的稳定性降低了约 3 千卡/摩尔，使蛋白质的稳定性略有降低，从而解释了其聚集行为。然而，对全局折叠动力学的影响仍然要小得多，转变态的稳定性降低小于 1 千卡/摩尔。值得注意的是，这种 1/3 转变态到折叠态的稳定性比为神秘的 Leffler α 值的分歧提供了一个明确的例子，这种分歧来自于环长度变化（通常为 1/3）和基于溶剂扰动的“标准”反应坐标（通常为>2/3）之间。为了解决这个问题，我们证明，当考虑到沿折叠途径发生的渐进环缩短时，分歧就会消失。该方法假设在平衡和动力学数据中 Flory 环熵标度因子 c = 1.48 是一致的，并具有验证由 phi 值分析确定的折叠核的三级相互作用的额外好处。因此，SOD1 不仅代表了一个关键催化功能的进化伴随着不稳定的 apo 状态的缺点的情况，而且还突出了作为探索蛋白质自组织的物理化学细节的理想模型系统。
