Bauer K, Struyvé M, Bosch D, Benz R, Tommassen J
Lehrstuhl für Biotechnologie der Universität Würzburg, Federal Republic of Germany.
J Biol Chem. 1989 Oct 5;264(28):16393-8.
Site-directed mutagenesis was performed with the phosphate starvation-inducible outer membrane porin PhoE of Escherichia coli K-12 to study the molecular basis of its anion selectivity. Lysines 18, 29, 64, and 125 were replaced by glutamic acids, and the properties of the mutant porins were investigated in in vivo and in vitro experiments. Lipid bilayer experiments showed that all these mutations had no influence on the pore structure because PhoE and the mutants had the same single channel conductance in KCl solution. Selectivity measurements revealed that the mutations changed the ionic selectivity of PhoE, but the change was dependent on the location of the lysine. Replacement of Lys18 and Lys29 by glutamic acid had a relatively small influence. The effect of the Lys64 substitution was somewhat larger, and the effect of the replacement of Lys125 resulted in the most drastic change in selectivity and in the loss of the interaction of PhoE with polyphosphate, whereas the replacement of the other lysines had no effect on the polyphosphate interaction behavior. The results are consistent with the assumption that the charge spot in PhoE consists of only 1 lysine per monomer, located in position 125 of the primary sequence and probably close to the pore interior.
利用大肠杆菌K-12的磷酸盐饥饿诱导型外膜孔蛋白PhoE进行定点诱变，以研究其阴离子选择性的分子基础。将赖氨酸18、29、64和125替换为谷氨酸，并在体内和体外实验中研究突变孔蛋白的性质。脂质双层实验表明，所有这些突变对孔结构均无影响，因为PhoE和突变体在KCl溶液中具有相同的单通道电导。选择性测量显示，这些突变改变了PhoE的离子选择性，但这种变化取决于赖氨酸的位置。用谷氨酸替换赖氨酸18和赖氨酸29的影响相对较小。赖氨酸64替换的影响稍大一些，而赖氨酸125替换的影响导致选择性变化最为剧烈，且PhoE与多磷酸盐的相互作用丧失，而其他赖氨酸的替换对多磷酸盐相互作用行为没有影响。这些结果与以下假设一致：PhoE中的电荷位点每个单体仅由一个赖氨酸组成，位于一级序列的第125位，可能靠近孔内部。
