The critical role of N- and C-terminal contact in protein stability and folding of a family 10 xylanase under extreme conditions.
The critical role of N- and C-terminal contact in protein stability and folding of a family 10 xylanase under extreme conditions.
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DOI：
10.1371/journal.pone.0011347
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发表时间：
2010-06-28
期刊：
影响因子：
3.7
通讯作者：
Reddy VS
中科院分区：
文献类型：
--
作者：
Bhardwaj A;Leelavathi S;Mazumdar-Leighton S;Ghosh A;Ramakumar S;Reddy VS
蛋白质在极端条件下所采取的稳定策略非常复杂，涉及各种相互作用。最近的研究表明，大部分蛋白质的N-和C-末端元件紧密接触，并表明它们在蛋白质折叠和稳定性中起作用。然而，这种接触的生物学意义仍然难以捉摸。在本研究中，我们调查的N-和C-末端残基的相互作用，使用家庭10木聚糖酶（BSX）与TIM桶结构，在高温下，碱性pH值，蛋白酶和SDS处理显示稳定性的作用。基于晶体结构，确定了一个芳香族簇，涉及Phe 4，Trp 6和Tyr 343将N-和C-末端保持在一起;这是该蛋白质的独特而重要的特征，可能对在多极端条件下的折叠和稳定性至关重要。创建了一系列突变体来破坏这种芳香族簇的形成，并研究在给定条件下稳定性和功能的丧失。虽然Phe 4的缺失导致稳定性丧失，但Trp 6和Tyr 343的去除影响体内折叠和活性。丙氨酸取代Phe 4，Trp 6和Tyr 343急剧下降的稳定性下的所有参数的研究。重要的是，用Trp取代Phe 4增加了SDS处理中的稳定性。有限蛋白水解的质谱结果进一步证明，Arg 344残基在敏感突变体如ΔF4、W 6A和Y343 A中对胰蛋白酶消化高度敏感，再次表明Phe 4-Trp 6-Tyr 343（F-W-Y）簇的破坏使N-和C-末端相互作用不稳定。我们的研究结果强调了通过芳香相互作用在蛋白质折叠和极端条件下的稳定性中的N-和C-末端接触的重要性，这些结果可能有助于改善其他蛋白质在次优条件下的稳定性。
Stabilization strategies adopted by proteins under extreme conditions are very complex and involve various kinds of interactions. Recent studies have shown that a large proportion of proteins have their N- and C-terminal elements in close contact and suggested they play a role in protein folding and stability. However, the biological significance of this contact remains elusive. In the present study, we investigate the role of N- and C-terminal residue interaction using a family 10 xylanase (BSX) with a TIM-barrel structure that shows stability under high temperature, alkali pH, and protease and SDS treatment. Based on crystal structure, an aromatic cluster was identified that involves Phe4, Trp6 and Tyr343 holding the N- and C-terminus together; this is a unique and important feature of this protein that might be crucial for folding and stability under poly-extreme conditions. A series of mutants was created to disrupt this aromatic cluster formation and study the loss of stability and function under given conditions. While the deletions of Phe4 resulted in loss of stability, removal of Trp6 and Tyr343 affected in vivo folding and activity. Alanine substitution with Phe4, Trp6 and Tyr343 drastically decreased stability under all parameters studied. Importantly, substitution of Phe4 with Trp increased stability in SDS treatment. Mass spectrometry results of limited proteolysis further demonstrated that the Arg344 residue is highly susceptible to trypsin digestion in sensitive mutants such as ΔF4, W6A and Y343A, suggesting again that disruption of the Phe4-Trp6-Tyr343 (F-W-Y) cluster destabilizes the N- and C-terminal interaction. Our results underscore the importance of N- and C-terminal contact through aromatic interactions in protein folding and stability under extreme conditions, and these results may be useful to improve the stability of other proteins under suboptimal conditions.
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影响因子：
56.9
作者：
LUGER, K;HOMMEL, U;KIRSCHNER, K
通讯作者：
KIRSCHNER, K
影响因子：
5.8
作者：
Dosztányi, Z;Magyar, C;Simon, I
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影响因子：
56.9
作者：
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通讯作者：
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DOI：
10.1073/pnas.0409114102
发表时间：
2005-01-25
影响因子：
11.1
作者：
Krishna, MMG;Englander, SW
通讯作者：
Englander, SW
影响因子：
2.9
作者：
LESK, AM;BRANDEN, CI;CHOTHIA, C
通讯作者：
CHOTHIA, C