Kobayashi Y, Nakae T
Eur J Biochem. 1985 Sep 2;151(2):231-6. doi: 10.1111/j.1432-1033.1985.tb09093.x.
The OmpF porin from the outer membrane of Escherichia coli acts as a lightly cation-selective pore, allowing the diffusion of small cations and cationic molecules, whose Mr are a little larger than the threshold exclusion limit. To ascertain the mechanism of this cation selectivity, we have examined a possible influence of cationic solutes on the fluorescence emission and the circular dichroic spectrum of tryptophan residues of the porin trimer, searching for conformational change(s). The diffusion of cationic solutes was determined with the native and the amidated porins in the presence or the absence of the effector cations. The following results were obtained. (a) Cations, e.g. spermidine, caused fluorescence quenching in the native trimer, with a half-maximum fluorescence quenching at 11-18 microM. A change in the circular dichroic spectrum was also recorded at around 280 nm. (b) The dissociation constant of spermidine to the native trimer was calculated to be 16 microM as determined by the method of equilibrium dialysis. (c) The cation-caused fluorescence quenching was reversed when the carboxyl groups of the trimer were modified by the amidation reaction, though amidation of the trimer resulted in no significant change in the fluorescence intensity. (d) The diffusion rate of N-benzyloxycarbonyl-glycyl-L-prolyl-L-arginine p-nitroanilide through the native and the amidated porins was lowered in the presence and the absence, respectively, of cations. Both the extent of fluorescence quenching in the presence of cation and the rate of cation diffusion were inversely proportional to the number of amidated carboxyl residues. The relative fluorescence quenching of the porin trimer (the amidated versus the native) in the presence of cations was linearly related to the relative solute diffusion via the porin (the amidated versus the native). These results suggested that cations caused a conformational change in the trimer, resulting in an easier diffusion of the solutes. The results suggested further that a limited number of carboxyl groups in the pore interior are involved in the cation selectivity of OmpF-porin pores.
来自大肠杆菌外膜的OmpF孔蛋白起着轻度阳离子选择性孔的作用，允许小阳离子和阳离子分子扩散，这些分子的相对分子质量略大于阈值排斥极限。为了确定这种阳离子选择性的机制，我们研究了阳离子溶质对孔蛋白三聚体中色氨酸残基的荧光发射和圆二色光谱的可能影响，以寻找构象变化。在有或没有效应阳离子存在的情况下，用天然和酰胺化的孔蛋白测定阳离子溶质的扩散。得到了以下结果。(a)阳离子，如亚精胺，导致天然三聚体中的荧光猝灭，在1 μM时荧光猝灭达到半最大值。在280 nm左右也记录到了圆二色光谱的变化。(b)通过平衡透析法测定，亚精胺与天然三聚体的解离常数为16 μM。(c)当三聚体的羧基通过酰胺化反应修饰时，阳离子引起的荧光猝灭被逆转，尽管三聚体的酰胺化导致荧光强度没有显著变化。(d)在有和没有阳离子存在的情况下，N-苄氧羰基-甘氨酰-L-脯氨酰-L-精氨酸对硝基苯胺通过天然和酰胺化孔蛋白的扩散速率分别降低。阳离子存在下的荧光猝灭程度和阳离子扩散速率均与酰胺化羧基残基的数量成反比。在阳离子存在下，孔蛋白三聚体（酰胺化与天然）的相对荧光猝灭与溶质通过孔蛋白的相对扩散（酰胺化与天然）呈线性相关。这些结果表明，阳离子导致三聚体发生构象变化，从而使溶质更容易扩散。结果还表明，孔内部有限数量的羧基参与了OmpF孔蛋白孔的阳离子选择性。
