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首页> 外文期刊>Journal of Molecular Biology >Overcoming hysteresis to attain reversible equilibrium folding for outer membrane phospholipase A in phospholipid bilayers.
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Overcoming hysteresis to attain reversible equilibrium folding for outer membrane phospholipase A in phospholipid bilayers.

机译：克服迟滞，使磷脂双层中的外膜磷脂酶A达到可逆的平衡折叠。

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摘要

The free energy of unfolding of a membrane protein from lipids into water (DeltaG(o)(w,l)) describes its equilibrium thermodynamic stability. Knowing this parameter gives insight into a membrane protein's sequence-structure-energy relationships. However, there are few measures of membrane protein stability because of the technical difficulties associated with unfolded and partially folded states. Here, we describe the experimental process that allowed us to measure the DeltaG(o)(w,l) of the outer membrane phospholipase A into large unilamellar vesicles (LUVs) of 1,2-dilauroyl-sn-glycero-3-phosphocholine. To arrive at this reversible folding condition, we screened a large number of experimental variables: temperature, incubation time, salt concentration, pH, lipid composition and liposome morphology. The principal challenge we encountered under most conditions was hysteresis between folding and unfolding titrations. A second factor that compromised reversible folding was the observation that a fraction of the protein population tended to aggregate. We found that hysteresis could be completely eliminated on a feasible timescale by conducting experiments at acidic pH, by the slow dilution of the protein in the initial titration setup and by utilizing a low concentration of a detergent as a temporary "holdase" to solubilize the protein upon its initial dilution into folding conditions. We confirmed that the detergent did not disrupt the LUVs using fluorescence emission of lipid-sensitive dyes and light scattering. The results of our parameter search should be generally useful for efforts to measure DeltaG(o)(w,l) for other membrane proteins.
机译：膜蛋白从脂质到水的展开自由能（DeltaG（o）（w，l））描述了其平衡热力学稳定性。知道此参数可以深入了解膜蛋白的序列-结构-能量关系。然而，由于与未折叠和部分折叠状态相关的技术困难，几乎没有膜蛋白稳定性的量度。在这里，我们描述了实验过程，该过程使我们能够将外膜磷脂酶A的DeltaG（o）（w，l）测量为1,2-二聚十二烷基-sn-甘油-3-磷酸胆碱的大单层囊泡（LUV）。为了达到这种可逆的折叠条件，我们筛选了大量实验变量：温度，孵育时间，盐浓度，pH，脂质组成和脂质体形态。在大多数条件下，我们遇到的主要挑战是折叠和展开滴定之间的滞后现象。损害可逆折叠的第二个因素是观察到一部分蛋白质群体倾向于聚集。我们发现通过在酸性pH下进行实验，通过在初始滴定装置中缓慢稀释蛋白质并利用低浓度的去污剂作为暂时的“保持酶”来溶解蛋白质，可以在可行的时间范围内完全消除滞后现象。在最初稀释成折叠条件时。我们证实，使用脂质敏感染料的荧光发射和光散射，该洗涤剂不会破坏LUV。我们的参数搜索结果通常应有助于测量其他膜蛋白的DeltaG（o）（w，l）。

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