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首页> 外文期刊>Biochemistry >Mapping of the residues involved in a proposed beta-strand located in the ferric enterobactin receptor FepA using site-directed spin-labeling
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Mapping of the residues involved in a proposed beta-strand located in the ferric enterobactin receptor FepA using site-directed spin-labeling

机译：使用定点自旋标记定位位于肠铁动蛋白受体FepA中的拟议β链中涉及的残基

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摘要

Electron paramagnetic resonance (EPR) site-directed spin-labeling (SDSL) has been used to characterize a proposed transmembrane beta-strand of the Escherichia coli ferric enterobactin receptor, FepA. Each of nine consecutive residues was mutated to cysteine and subsequently labeled with the sulfhydryl-specific spin-label methanethiosulfonate (MTSL) and the purified protein reconstituted into liposomes. Continuous wave (CW) power saturation methods were used to determine exposure of the nitroxide side chains to a series of paramagnetic relaxation agents, including nickel acetylacetonate (NiAA), nickel ethylenediaminediacetate (NiEDDA), chromium oxalate (CROX), and molecular oxygen. The spin-label attached to Q245C, L247C, L249C, A251C, and Y253C had higher collision frequencies with molecular oxygen than with polar relaxation agents, indicating that these sites are exposed to the hydrophobic phase of the lipid bilayer. MTSL bound to residues S246C, E248C, E250C, and G252C had higher collision rates with the polar agents than with oxygen, suggesting that these sites are exposed to the aqueous channel. The alternating periodicity observed with the polar relaxation agents, NiAA and NiEDDA, and in opposite phase with oxygen, is consistent with beta-sheet structure. Depth measurements, based on the reciprocal concentration gradients of NiEDDA and O2 across the bilayer and calibrated for our system with phosphatidylcholine spin-labels, indicated that L249C was nearest the center of the bilayer and that Q245C and Y253C were located just below the bilayer surface in opposite leaflets of the membrane. Thus, we conclude that this approach, through mapping of individual residues, has the capability of defining beta-sheet secondary structure.
机译：电子顺磁共振（EPR）定点自旋标记（SDSL）已用于表征大肠杆菌铁肠杆菌素受体FepA的拟议跨膜β链。将九个连续残基中的每一个突变为半胱氨酸，随后用巯基特异性自旋标记的甲硫基磺酸盐（MTSL）进行标记，并将纯化的蛋白质重构为脂质体。连续波（CW）功率饱和方法用于确定一氧化氮侧链暴露于一系列顺磁性弛豫剂，包括乙酰丙酮镍（NiAA），乙二胺二乙酸镍（NiEDDA），草酸铬（CROX）和分子氧。附着在Q245C，L247C，L249C，A251C和Y253C上的自旋标记与分子氧的碰撞频率高于与极性松弛剂的碰撞频率，表明这些位点暴露于脂质双层的疏水相。与残基S246C，E248C，E250C和G252C结合的MTSL与极性试剂的碰撞速率高于与氧气的碰撞速率，表明这些位点暴露于水通道。用极性弛豫剂NiAA和NiEDDA观察到的交替周期，以及与氧相反的相，与β-折叠结构一致。根据NiEDDA和O2在整个双层上的倒数浓度梯度并针对我们的系统使用磷脂酰胆碱自旋标记物进行校准，进行深度测量，结果表明L249C最靠近双层中心，而Q245C和Y253C恰好位于双层的下方。膜的相对小叶。因此，我们得出结论，该方法通过绘制单个残基，具有定义β-折叠二级结构的能力。

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