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首页> 外文期刊>Biochemistry >Direct Detection of Hydrogen Bonds in Monomeric Superoxide Dismutase: Biological Implications
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Direct Detection of Hydrogen Bonds in Monomeric Superoxide Dismutase: Biological Implications

机译：直接检测单体超氧化物歧化酶中的氢键：生物学意义。

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Hydrogen bonds were directly determined via NMR with different experimental approaches at 600 and 800 MHz for reduced monomeric superoxide dismutase (Q133M2SOD, 16 kDa). This protein contains a copper and a zinc ion and shows the classical superoxide dismutase (SOD) eight-stranded #beta#-barrel fold. The best results for this intermediate molecular mass protein were obtained using a TROSY version of the long-range HNCO experiment at high magnetic field (800 MHz) or with a cryoprobe at 600 MHz. The backbone hydrogen bond network that defines the secondary structure of the protein was detected. Thirty-five backbone hydrogen bonds were identified. The lower limit for their detection, their relation to the TROSY R_2 rates, and the correlation between hydrogen bond detectability and signal line width are discussed. Experiments were also optimized to detect hydrogen bonds involving key side chains, which lead to the observation of five hydrogen bonds. In paricular, the hydrogen bonds involving the side chains of Asp 124 were observed, which show significant differences with respect to the bonds expected on the basis of the crystal structure. The relevance of this finding relies also on the fact that Asp 124 is a key residue in determining the affinity of the protein for zinc. It has now been determined that the gain of the toxic function of peroxynitrite formation in SOD mutants related to amyotrophic lateral sclerosis (ALS) is due to SOD species lacking the zinc the zinc ion, as a consequence of reduced affinity for zinc. Therefore, this study provides structural hints for understanding the origin of the enzymatic behavior of the Zn-deficient SOD.
机译：氢键直接通过NMR用不同的实验方法在600和800 MHz上确定还原的单体超氧化物歧化酶（Q133M2SOD，16 kDa）。该蛋白质包含铜和锌离子，显示出经典的超氧化物歧化酶（SOD）八链＃beta＃-桶状折叠。使用TROSY版本的远距离HNCO实验在高磁场（800 MHz）下或在600 MHz的低温探头下，可获得此中等分子量蛋白质的最佳结果。检测到定义蛋白质二级结构的主链氢键网络。鉴定了35个主链氢键。讨论了它们的检测下限，它们与TROSY R_2速率的关系以及氢键可检测性与信号线宽度之间的相关性。还对实验进行了优化，以检测涉及关键侧链的氢键，从而观察到五个氢键。特别地，观察到涉及Asp 124侧链的氢键，其相对于根据晶体结构预期的键显示出显着差异。该发现的相关性还取决于Asp 124是确定蛋白质与锌亲和力的关键残基这一事实。现在已经确定，与肌萎缩性侧索硬化症（ALS）有关的SOD突变体中过亚​​硝酸盐形成的毒性功能的增加是由于SOD种类缺乏锌，锌离子，这是由于对锌的亲和力降低。因此，本研究为理解缺锌SOD的酶促行为的起源提供了结构性提示。

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