Protein-lipid interactions with Fusobacterium nucleatum major outer membrane protein FomA:: Spin-label EPR and polarized infrared spectroscopy
Protein-lipid interactions with Fusobacterium nucleatum major outer membrane protein FomA:: Spin-label EPR and polarized infrared spectroscopy
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DOI：
10.1021/bi800750s
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发表时间：
2008-08-12
期刊：
影响因子：
2.9
通讯作者：
Marsh, D.
中科院分区：
文献类型：
--
作者：
Anbazhagan, V.;Vijay, N.;Marsh, D.
在大肠杆菌中表达和纯化了核梭杆菌的主要外膜蛋白FOMA，并用洗涤剂在C(12：0)到C(17：0)的磷脂酰胆碱双层膜中重组。通过偏振、衰减全反射、红外光谱和自旋标记脂类的电子顺磁共振(EPR)谱表征了FOMA与膜结合蛋白的构象和取向。通过IR谱带拟合，估计约190个膜性FOMA残基处于β-折叠构型，这与14链跨膜β-桶结构相一致。FOMA的IR二色性分析表明，β链相对于薄片/桶轴倾斜约45度，后者的有序参数表现出与流体C(13：0)脂链疏水匹配的不连续性。EPR测得的脂-蛋白相互作用的化学计量比(N(B)=23脂/单体)表明，FOMA在DIC(14：0)磷脂酰胆碱膜中不是三聚体，与14-16链的单体β-桶一致。与阴离子自旋标记脂类相互作用的显著选择性将蛋白质的碱性残基放置在极性-非极膜界面附近，与当前的拓扑模型一致。与8-22链的大肠杆菌外膜蛋白OmpA、OmpG和FhuA的相似数据比较，支持上述结论。
FomA, the major outer membrane protein of Fusobacterium nucleatum, was expressed and purified in Escherichia coli and reconstituted from detergent in bilayer membranes of phosphatidylcholines with chain lengths from C(12:0) to C(17:0). The conformation and orientation of membrane-incorporated FomA were determined from polarized, attenuated total reflection, infrared (IR) spectroscopy, and lipid-protein interactions with FomA were characterized by using electron paramagnetic resonance (EPR) spectroscopy of spin-labeled lipids. Approximately 190 residues of membranous FomA are estimated to be in a beta-sheet configuration from IR band fitting, which is consistent with a 14-strand transmembrane beta-barrel structure. IR dichroism of FomA indicates that the beta-strands are tilted by similar to 45 degrees relative to the sheet/barrel axis and that the order parameter of the latter displays a discontinuity corresponding to hydrophobic matching with fluid C(13:0) lipid chains. The stoichiometry (N(b) = 23 lipids/monomer) of lipid-protein interaction from EPR demonstrates that FomA is not trimeric in membranes of diC(14:0) phosphatidylcholine and is consistent with a monomeric beta-barrel of 14-16 strands. The pronounced selectivity of interaction found with anionic spin-labeled lipids places basic residues of the protein in the vicinity of the polar-apolar membrane interfaces, consistent with current topology models. Comparison with similar data from the 8- to 22-stranded E. coli outer membrane proteins, OmpA, OmpG, and FhuA, supports the above conclusions.