Effects of glycosylation on the stability and flexibility of a metastable protein: the human serpin α(1)-antitrypsin.
Effects of glycosylation on the stability and flexibility of a metastable protein: the human serpin α(1)-antitrypsin.
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DOI：
10.1016/j.ijms.2010.08.003
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发表时间：
2011-04
影响因子：
1.8
通讯作者：
Wintrode PL
中科院分区：
文献类型：
--
作者：
Sarkar A;Wintrode PL
蛋白质糖基化通常稳定蛋白质，从而增加蛋白质半衰期并防止变性或蛋白水解降解。虽然通常是有益的，但在抑制性丝氨酸蛋白酶抑制剂的情况下，这种稳定化可能是不利的。这些蛋白酶抑制剂是亚稳态的，并且向更稳定形式的构象转变是其功能的关键。因此，不稳定性对于这些抑制性丝氨酸蛋白酶抑制剂是必不可少的，并且诱变已经证明实质性稳定导致功能受损。我们用光谱、氢/氘交换和质谱研究了糖基化对人丝氨酸蛋白酶抑制剂α 1-抗胰蛋白酶（α1-AT）的影响。先前的研究发现，未糖基化的重组α1-AT在低变性剂下填充熔融球，并且填充这种状态的能力与有效的蛋白酶抑制相关。此外，在几个重要区域发现了高度的构象灵活性。通过圆二色性监测盐酸胍变性，表明血浆α1-AT（在3个位点糖基化）相对于非糖基化形式基本稳定。然而，氢交换显示高于1 M GuHCl的血浆α1-AT保护作用完全丧失，与重组形式相似。因此，糖似乎稳定了α1-AT的紧密变性状态，而没有显著稳定折叠状态。天然状态的氢交换揭示了天然的灵活性的微小扰动，但在关键区域，如f螺旋的高灵活性是保守的。β-链1c在血浆α1-AT中稳定，这可能增加对形成致病性聚合物的抗性。总的来说，我们的研究结果表明，抑制性丝氨酸蛋白酶抑制剂的糖基化不干扰天然状态的灵活性或天然的不稳定性，这是有效的功能所需的，虽然它可能会赋予抵抗蛋白酶降解，从而延长循环丝氨酸蛋白酶抑制剂的半衰期。
Protein glycosylation commonly stabilizes proteins thereby increasing protein half-lives and protecting against denaturation or proteolytic degradation. While generally beneficial, such stabilization is potentially disadvantageous in the case of inhibitory serpins. These protease inhibitors are metastable and a conformational transition to a more stable form is key to their function. Instability is therefore essential for these inhibitory serpins and mutagenesis has demonstrated that substantial stabilization results in compromised function. We have used optical spectroscopy and hydrogen/deuterium exchange and mass spectrometry to investigate the effects of glycosylation on the human serpin alpha-1 antitrypsin (α1-AT). Previous studies found that unglycosylated recombinant α1-AT populates a molten globule at low denaturant and that the ability to populate this state is correlated with efficient protease inhibition. Further, a high degree of conformational flexibility was found in several important regions. Guanidine hydrochloride denaturation monitored by circular dichroism indicates that plasma α1-AT, which is glycosylated at 3 sites, is substantially stabilized relative to the unglycosylated form. However, hydrogen exchange reveals complete loss of protection in plasma α1-AT above 1 M GuHCl, similar to what is seen for the recombinant form. Sugars therefore appear to stabilize the compact denatured state of α1-AT without significant stabilization of the folded state. Native state hydrogen exchange reveals minor perturbations to native flexibility, but high flexibility in key regions such as the f helix is conserved. β-strand 1c is stabilized in plasma α1-AT, which may confer increased resistance to forming pathogenic polymers. Overall, our results indicate that glycosylation of inhibitory serpins does not interfere with either native state flexibility or the native instability that is required for efficient function, though it may confer resistance to degradation by proteases and thus extend the half-life of circulating serpins.
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DOI：
10.1038/nsb1101-979
发表时间：
2001-11-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
Ye, S;Cech, AL;Goldsmith, EJ
通讯作者：
Goldsmith, EJ
影响因子：
2.9
作者：
Cabrita, LD;Whisstock, JC;Bottomley, SP
通讯作者：
Bottomley, SP
影响因子：
8
作者：
Elliott, PR;Pei, XY;Lomas, DA
通讯作者：
Lomas, DA
影响因子：
14.9
作者：
Bohne-Lang A;von der Lieth CW
通讯作者：
von der Lieth CW
DOI：
10.1016/s0304-4165(96)00143-2
发表时间：
1997-06-06
影响因子：
3
作者：
Kwon, KS;Yu, MH
通讯作者：
Yu, MH