Factors influencing the solubilization of membrane proteins from Escherichia coli membranes by styrene-maleic acid copolymers
Factors influencing the solubilization of membrane proteins from Escherichia coli membranes by styrene-maleic acid copolymers
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DOI：
10.1016/j.bbamem.2019.183125
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发表时间：
2020-02-01
影响因子：
3.4
通讯作者：
Killian, J. Antoinette
中科院分区：
文献类型：
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作者：
Kopf, Adrian H.;Dorr, Jonas M.;Killian, J. Antoinette
苯乙烯-马来酸（SMA）共聚物是一种很有前途的替代去污剂的膜蛋白的增溶。在这里，我们采用大肠杆菌膜含有KcsA作为模型蛋白，研究不同的环境条件对SMA的增溶效率的影响。我们表明，SMA浓度，温度，孵育时间，离子强度，二价阳离子的存在和pH值都影响SMA提取的蛋白质的量。所观察到的效果是一致的，从脂质模型膜系统的观察，除了pH值的影响。增加pH值从7到9被发现，导致在增加的增溶产率的E。colt膜，而在仅脂质模型系统中，它在相同的pH范围内下降，基于光密度（OD）测量。在比较天然酵母膜和仅酵母脂质膜的溶解的OD实验中观察到类似的相反pH依赖性效应。我们提出了一个模型，其中pH值依赖性的静电相互作用影响的聚合物的膜蛋白的膜外部分的结合，这反过来又影响膜溶解的聚合物的可用性。该模型得到以下观察结果的支持：对于阴离子洗涤剂SDS观察到与SMA类似的pH依赖性，但对于非离子洗涤剂DDM则没有，并且pH依赖性可以通过增加SMA浓度而在很大程度上克服。结果是有用的指导方针，以获得最佳的条件下溶解的生物膜SMA。
Styrene-maleic acid (SMA) copolymers are a promising alternative to detergents for the solubilization of membrane proteins. Here we employ Escherichia colt membranes containing KcsA as a model protein to investigate the influence of different environmental conditions on SMA solubilization efficiency. We show that SMA concentration, temperature, incubation time, ionic strength, presence of divalent cations and pH all influence the amount of protein that is extracted by SMA. The observed effects are consistent with observations from lipid-only model membrane systems, with the exception of the effect of pH. Increasing pH from 7 to 9 was found to result in an increase of the solubilization yield of E. colt membranes, whereas in lipid-only model systems it decreased over the same pH range, based on optical density (OD) measurements. Similar opposite pH dependent effects were observed in OD experiments comparing solubilization of native yeast membranes and yeast lipid-only membranes. We propose a model in which pH-dependent electrostatic interactions affect binding of the polymers to extramembraneous parts of membrane proteins, which in turn affects the availability of polymer for membrane solubilization. This model is supported by the observations that a similar pH-dependence as for SMA is observed for the anionic detergent SDS, but not for the nonionic detergent DDM and that the pH-dependence can be largely overcome by increasing the SMA concentration. The results are useful as guidelines to derive optimal conditions for solubilization of biological membranes by SMA.