Mutation of the critical pH-gating residues histidine 231 to glutamate increase open probability of outer membrane protein G in planar lipid bilayer
Mutation of the critical pH-gating residues histidine 231 to glutamate increase open probability of outer membrane protein G in planar lipid bilayer
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DOI：
10.1007/s13238-013-3070-5
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发表时间：
2013-09
期刊：
影响因子：
21.1
通讯作者：
Mu Yu;Peibei Sun;Yao He;Liang Xiao;Demeng Sun;Long-hua Zhang;C. Tian
中科院分区：
文献类型：
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作者：
Mu Yu;Peibei Sun;Yao He;Liang Xiao;Demeng Sun;Long-hua Zhang;C. Tian
外膜蛋白G（OmpG）是大肠杆菌（Escherichia coli，E. coli），允许高达900 Da的离子和分子通过（法哈尔多等人，1998年）。它由280个氨基酸组成，形成14链β-折叠，在细胞外侧具有7个长环（L1-L7），在周质侧具有6个短转角（Subbarao and货车den贝格，2006; Yildiz et al.，2006; Liang和Tamm，2007）。尽管OmpG基因存在于几种大肠杆菌的基因组中，但OmpG基因在大肠杆菌中的表达量并不高。coli菌株（Nikaido，1999）中，OmpG的表达仅在E.缺乏OmpF和LamB的大肠杆菌突变体（法哈尔多等人，1998），以使麦芽糖糊精能够扩散穿过细菌外膜。非常有趣的是，与通常的三聚体通道形成孔蛋白不同，OmpG在生理和结构研究中表现出功能单体的迷人特征（Conlan和Bayley，2003; Mari等人，2010年）。最近的原子力显微镜（AFM）研究显示了脂质中的二聚OmpG，但没有证据显示OmpG的寡聚形式的生理相关性（Mari等人，2010年）。功能性单体通道孔蛋白使得OmpG能够被工程化（Bayley等人，2008）作为单分子生物传感器（Chen等人，2008年a）。OmpG在平面脂质双层中的体外研究揭示OmpG的通道性质是pH依赖性、电压依赖性和自发门控性的（Conlan等人，2000; Conlan and Bayley，2003）.通道蛋白质的高分辨率晶体记录。功能单体OmpG自然是随机传感的最佳候选者，尽管其pH依赖性门控。几个突变被工程化以减弱或消除OmpG的pH门控特性，例如降低环L 6移动性（在链β12和β13的细胞外末端之间引入二硫键）或优化β11和β12之间的链内氢键（缺失Asp 215）（Chen et al.，2008年a、2008年b）。为了进一步降低OmpG通道的pH依赖性门控，在这项工作中，pH敏感的His 231残基被带负电荷的残基谷氨酸（OmpG-H231 E）取代。结果表明，OmpG-WT和OmpG-H231 E均以包涵体的形式表达，OmpG-WT和OmpG-H231 E均以包涵体的形式表达。纯化的OmpG蛋白使用高浓度的尿素充分溶解并在DM胶束中纯化（图S1）。采用透析法对OmpG蛋白在POPE/POPG（3：1）脂质体中进行去污剂介导的重构。在蛋白脂质体扩散到平面脂质双层装置的预涂孔后，观察到的通道电流表明OmpG蛋白在脂质体中良好折叠（图1），表明脂质介导的蛋白质重折叠用于通道电导研究的可靠方法。
Outer-membrane protein G (OmpG) is a nonspecific β-barrel porin in the outer membrane of Escherichia coli (E. coli), allowing the passage of ions and molecules up to 900 Da (Fajardo et al., 1998). It comprises of 280 amino acids that form 14-stranded β-sheets with seven long loops (L1–L7) on the extracellular side and six short turns on the periplasmic side (Subbarao and van den Berg, 2006; Yildiz et al., 2006; Liang and Tamm, 2007). Despite that the OmpG gene exists in the genome of several E. coli strains (Nikaido, 1999), expression of OmpG was only observed in E. coli mutants lacking OmpF and LamB (Fajardo et al., 1998) to enable the diffusion of maltodextrins across the bacterial outer membrane. Very interestingly, unlike usual trimeric channel-forming porins, OmpG exhibits fascinating characteristics of a functional monomer in physiological and structural studies (Conlan and Bayley, 2003; Mari et al., 2010). Recent atomic force microscopy (AFM) studies showed the dimeric OmpG in lipid, but no evidence shows the physiological relevance of oligomeric forms of OmpG (Mari et al., 2010). The functional monomeric channel porin enables OmpG to be engineered (Bayley et al., 2008) as a single-molecule biosensor (Chen et al., 2008a). The in vitro studies of OmpG in planar lipid bilayers revealed that channel properties of OmpG were pH-dependent, voltage-dependent, and spontaneous gating (Conlan et al., 2000; Conlan and Bayley, 2003). High resolution crysrecording of channel proteins. The functional monomeric OmpG is naturally the best candidate for stochastic sensing, despite of its pH-dependent gating. Several mutations were engineered to weaken or abolish the pH gating properties of OmpG, such as reducing loop L6 mobility (introducing a disulfide bond between the extracellular ends of strands β12 and β13) or optimizing intrastrand hydrogen bonding between β11 and β12 (deletion of Asp 215)(Chen et al., 2008a, 2008b). To further reduce the pH-dependent gating of the OmpG channel for a quiet channel biosensor, the pH sensitive His 231 residue was replaced by a negatively charged residue glutamate (OmpG-H231E) in this work. The charge interaction between Glu 231 and His 261 was proposed to stabilize the upright fold of L6 (especially at acidic pH).Using the bacterial expression system, both OmpG-WT and OmpG-H231E were over-expressed as inclusion bodies. The purified OmpG proteins were well solubilized using high concentration of urea and purified in DM micelles (Fig. S1). The detergent mediated reconstitution of OmpG proteins in POPE/POPG (3: 1) liposomes was conducted using the dialysis methods. After the proteoliposome was diffused to the prepainted aperture of planar lipid bilayer apparatus, the observed channel current demonstrated that the OmpG proteins were well folded in liposome (Fig. 1), indicating a reliable method of lipid mediated protein refolding for channel conductance studies.