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首页> 外文期刊>Journal of Molecular Biology >Reversible unfolding of a thermophilic membrane protein in phospholipid/detergent mixed micelles.
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Reversible unfolding of a thermophilic membrane protein in phospholipid/detergent mixed micelles.

机译：磷脂/洗涤剂混合胶束中嗜热膜蛋白的可逆展开。

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Folding mechanisms and stability of membrane proteins are poorly understood because of the known difficulties in finding experimental conditions under which reversible denaturation could be possible. In this work, we describe the equilibrium unfolding of Archaeoglobus fulgidus CopA, an 804-residue alpha-helical membrane protein that is involved in transporting Cu(+) throughout biological membranes. The incubation of CopA reconstituted in phospholipid/detergent mixed micelles with high concentrations of guanidinium hydrochloride induced a reversible decrease in fluorescence quantum yield, far-UV ellipticity, and loss of ATPase and phosphatase activities. Refolding of CopA from this unfolded state led to recovery of full biological activity and all the structural features of the native enzyme. CopA unfolding showed typical characteristics of a two-state process, with DeltaG(w) degrees =12.9 kJ mol(-)(1), m=4.1 kJ mol(-1) M(-1), C(m)=3 M, and DeltaCp(w) degrees =0.93 kJ mol(-1) K(-1). These results point out to a fine-tuning mechanism for improving protein stability. Circular dichroism spectroscopic analysis of the unfolded state shows that most of the secondary and tertiary structures were disrupted. The fraction of Trp fluorescence accessible to soluble quenchers shifted from 0.52 in the native state to 0.96 in the unfolded state, with a significant spectral redshift. Also, hydrophobic patches in CopA, mainly located in the transmembrane region, were disrupted as indicated by 1-anilino-naphtalene-8-sulfonate fluorescence. Nevertheless, the unfolded state had a small but detectable amount of residual structure, which might play a key role in both CopA folding and adaptation for working at high temperatures.
机译：膜蛋白的折叠机制和稳定性知之甚少，因为在寻找可能进行可逆变性的实验条件方面存在已知困难。在这项工作中，我们描述了古细菌fulgidus CopA的平衡展开，这是一个804残基的α-螺旋膜蛋白，参与在整个生物膜中运输Cu（+）。在具有高浓度盐酸胍的磷脂/洗涤剂混合胶束中重构的CopA的孵育导致荧光量子产率，远紫外椭圆率以及ATPase和磷酸酶活性的可逆降低。从该展开状态重新折叠CopA导致完全生物活性和天然酶的所有结构特征的恢复。 CopA展开显示了两个状态过程的典型特征，DeltaG（w）度= 12.9 kJ mol（-）（1），m = 4.1 kJ mol（-1）M（-1），C（m）= 3 M和DeltaCp（w）度= 0.93 kJ mol（-1）K（-1）。这些结果指出了改善蛋白质稳定性的微调机制。展开状态的圆二色光谱分析表明，大多数二级和三级结构均被破坏。可溶性淬灭剂可及的Trp荧光分数从天然状态的0.52变为未折叠状态的0.96，具有明显的光谱红移。而且，如1-苯胺基-萘-8-磺酸盐荧光所示，CopA中主要位于跨膜区的疏水性斑块被破坏。然而，展开状态具有少量但可检测到的残留结构，这可能在CopA折叠和适应高温工作中都起着关键作用。

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