The effects of a protein osmolyte on the stability of the integral membrane protein glycerol facilitator
The effects of a protein osmolyte on the stability of the integral membrane protein glycerol facilitator
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DOI：
10.1139/bcb-2014-0076
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发表时间：
2014-12-01
期刊：
影响因子：
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通讯作者：
O'Neil, Joe D.
中科院分区：
文献类型：
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作者：
Baturin, Simon;Galka, Jamie J.;O'Neil, Joe D.
渗透物是一种天然存在的分子，被各种各样的生物体用来在极端的温度、盐度、静水压力、变性剂浓度和干燥条件下稳定蛋白质。研究了渗透液三甲胺n -氧化物（TMAO）对大肠杆菌整体膜蛋白甘油促进剂（GF）四聚体热变性和十二烷基硫酸钠（SDS）化学变性稳定性的影响，以及洗剂头基团和酰基链长度的影响。TMAO促进正常四聚体α -螺旋蛋白与十二烷基麦芽糖苷（DDM）的高阶低聚物结合，但不促进十四烷基麦芽糖苷（TDM）、赖氨酸月桂酰磷脂酰胆碱（LLPC）或赖氨酸肉豆醇酰磷脂酰胆碱（LMPC）的结合，这是由动态光散射（DLS）确定的；如SDS聚丙烯酰胺凝胶电泳所示，八聚体复合物特别稳定。氧化三甲胺使GF四聚体的热稳定性在4种洗涤剂中平均提高了10℃，并保护蛋白质不被SDS变性。然而，当添加到sds解离蛋白中时，它并没有促进与四聚体的重新结合。氧化三甲胺还促进棒状洗涤剂胶束的形成，DLS被发现可用于监测蛋白质的结构和蛋白质热解离过程中洗涤剂的重新分配。该蛋白在具有磷脂酰胆碱头基团（LLPC和LMPC）的洗涤剂中比在麦芽糖苷洗涤剂中更热稳定。讨论了该结果对渗透机理、膜蛋白稳定性和蛋白-蛋白相互作用的影响。
Osmolytes are naturally occurring molecules used by a wide variety of organisms to stabilize proteins under extreme conditions of temperature, salinity, hydrostatic pressure, denaturant concentration, and desiccation. The effects of the osmolyte trimethylamine N-oxide (TMAO) as well as the influence of detergent head group and acyl chain length on the stability of the Escherichia coli integral membrane protein glycerol facilitator (GF) tetramer to thermal and chemical denaturation by sodium dodecyl sulphate (SDS) are reported. TMAO promotes the association of the normally tetrameric alpha-helical protein into higher order oligomers in dodecyl-maltoside (DDM), but not in tetradecyl-maltoside (TDM), lyso-lauroylphosphatidyl choline (LLPC), or lyso-myristoylphosphatidyl choline (LMPC), as determined by dynamic light scattering (DLS); an octameric complex is particularly stable as indicated by SDS polyacrylamide gel electrophoresis. TMAO increases the heat stability of the GF tetramer an average of 10 degrees C in the 4 detergents and also protects the protein from denaturation by SDS. However, it did not promote re-association to the tetramer when added to SDS-dissociated protein. TMAO also promotes the formation of rod-like detergent micelles, and DLS was found to be useful for monitoring the structure of the protein and the redistribution of detergent during thermal dissociation of the protein. The protein is more thermally stable in detergents with the phosphatidylcholine head group (LLPC and LMPC) than in the maltoside detergents. The implications of the results for osmolyte mechanism, membrane protein stability, and protein-protein interactions are discussed.