Doyle Colleen M, Rumfeldt Jessica A, Broom Helen R, Sekhar Ashok, Kay Lewis E, Meiering Elizabeth M
Program in Molecular Structure and Function, Hospital for Sick Children , Toronto, Canada.
Biochemistry. 2016 Mar 8;55(9):1346-61. doi: 10.1021/acs.biochem.5b01133. Epub 2016 Feb 22.
The chemical shifts of backbone amide protons in proteins are sensitive reporters of local structural stability and conformational heterogeneity, which can be determined from their readily measured linear and nonlinear temperature-dependences, respectively. Here we report analyses of amide proton temperature-dependences for native dimeric Cu, Zn superoxide dismutase (holo pWT SOD1) and structurally diverse mutant SOD1s associated with amyotrophic lateral sclerosis (ALS). Holo pWT SOD1 loses structure with temperature first at its periphery and, while having extremely high global stability, nevertheless exhibits extensive conformational heterogeneity, with ∼1 in 5 residues showing evidence for population of low energy alternative states. The holo G93A and E100G ALS mutants have moderately decreased global stability, whereas V148I is slightly stabilized. Comparison of the holo mutants as well as the marginally stable immature monomeric unmetalated and disulfide-reduced (apo(2SH)) pWT with holo pWT shows that changes in the local structural stability of individual amides vary greatly, with average changes corresponding to differences in global protein stability measured by differential scanning calorimetry. Mutants also exhibit altered conformational heterogeneity compared to pWT. Strikingly, substantial increases as well as decreases in local stability and conformational heterogeneity occur, in particular upon maturation and for G93A. Thus, the temperature-dependence of amide shifts for SOD1 variants is a rich source of information on the location and extent of perturbation of structure upon covalent changes and ligand binding. The implications for potential mechanisms of toxic misfolding of SOD1 in disease and for general aspects of protein energetics, including entropy-enthalpy compensation, are discussed.
蛋白质中主链酰胺质子的化学位移是局部结构稳定性和构象异质性的敏感报告物，它们可分别通过易于测量的线性和非线性温度依赖性来确定。在此，我们报告了对天然二聚体铜锌超氧化物歧化酶（全酶野生型SOD1）以及与肌萎缩侧索硬化症（ALS）相关的结构多样的突变型SOD1的酰胺质子温度依赖性分析。全酶野生型SOD1首先在其外围随温度失去结构，并且尽管具有极高的整体稳定性，但仍表现出广泛的构象异质性，约五分之一的残基显示出存在低能量替代状态的证据。全酶G93A和E100G ALS突变体的整体稳定性适度降低，而V148I则略有稳定。将全酶突变体以及边缘稳定的未成熟单体未金属化和二硫键还原（脱辅基（2SH））野生型与全酶野生型进行比较表明，各个酰胺局部结构稳定性的变化差异很大，平均变化对应于通过差示扫描量热法测量的蛋白质整体稳定性差异。与野生型相比，突变体还表现出构象异质性的改变。引人注目的是，局部稳定性和构象异质性出现了大幅增加以及降低的情况，特别是在成熟时以及对于G93A而言。因此，SOD1变体酰胺位移的温度依赖性是关于共价变化和配体结合后结构扰动的位置和程度的丰富信息来源。文中讨论了SOD1在疾病中有毒错误折叠的潜在机制以及蛋白质能量学的一般方面（包括熵 - 焓补偿）的意义。
