Qazi S Junaid S, Chew Raymond, Bay Denice C, Turner Raymond J
Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4.
Biochem Biophys Rep. 2015 Mar 26;1:22-32. doi: 10.1016/j.bbrep.2015.03.007. eCollection 2015 May.
EmrE is a member of the small multidrug resistance (SMR) protein family in . EmrE confers resistance to a wide variety of quaternary cation compounds (QCCs) as an efflux transporter driven by proton motive force. The purification yield of most membrane proteins are challenging because of difficulties in over expressing, isolating and solubilizing them and the addition of an affinity tag often improves purification. The purpose of this study is to compare the structure and function of hexahistidinyl (His) tagged (T-EmrE) and untagged (UT-EmrE) versions of EmrE. QCC resistance assays determined that T-EmrE demonstrated reduced resistance as compared to UT-EmrE. We isolated EmrE using the two different purification methods, an organic solvent extraction method used to isolate UT-EmrE and nickel affinity chromatography of T-EmrE. All proteins were solubilized in the same buffered n-dodecyl-β-d-maltopyranoside (DDM) detergent and their conformations were examined in the presence/absence of different QCCs. analysis of protein multimerization using SDS-Tricine PAGE and dynamic light scattering analysis revealed that both proteins predominated as monomers, but the formation of dimers was more constant and uniform in T-EmrE compared to UT-EmrE. The aromatic residue conformations of both proteins indicate that T-EmrE form is more aqueous exposed than UT-EmrE, but UT-EmrE appeared to have a more dynamic environment surrounding its aromatic residues. Using fluorescence to obtain QCC ligand-binding curves indicated that the two forms had differences in dissociation constants ( ) and maximum specific one-site binding ( ) values for particular QCCs. analyses of both proteins demonstrated subtle but significant differences in multimerization and QCC binding. analysis indicates differences caused by the addition of the tag, we also observed differences that could be a result of the tag and/or the different purification methods.
EmrE是[具体物种]中小多重耐药性（SMR）蛋白家族的成员。作为由质子动力驱动的外排转运蛋白，EmrE赋予对多种季铵阳离子化合物（QCCs）的抗性。由于膜蛋白在过量表达、分离和溶解方面存在困难，大多数膜蛋白的纯化产率具有挑战性，而添加亲和标签通常可提高纯化效果。本研究的目的是比较六组氨酸标签化（His-tagged，T-EmrE）和未标签化（untagged，UT-EmrE）的EmrE的结构和功能。QCC抗性测定表明，与UT-EmrE相比，T-EmrE的抗性降低。我们使用两种不同的纯化方法分离EmrE，一种用于分离UT-EmrE的有机溶剂萃取法和T-EmrE的镍亲和层析法。所有蛋白质均溶解于相同的缓冲正十二烷基-β-D-麦芽糖苷（DDM）洗涤剂中，并在存在/不存在不同QCCs的情况下检查其构象。使用SDS-三羟甲基氨基甲烷聚丙烯酰胺凝胶电泳（SDS-Tricine PAGE）分析蛋白质多聚化和动态光散射分析表明，两种蛋白质均以单体为主，但与UT-EmrE相比，T-EmrE中更稳定且均匀地形成二聚体。两种蛋白质的芳香族残基构象表明，T-EmrE形式比UT-EmrE更暴露于水环境中，但UT-EmrE在其芳香族残基周围似乎具有更动态的环境。使用荧光获得QCC配体结合曲线表明，两种形式在特定QCC的解离常数（）和最大特异性单点结合（）值方面存在差异。对两种蛋白质的分析表明，在多聚化和QCC结合方面存在细微但显著的差异。分析表明添加标签会导致差异，我们还观察到可能是标签和/或不同纯化方法导致的差异。
