Nestorovich Ekaterina M, Sugawara Etsuko, Nikaido Hiroshi, Bezrukov Sergey M
Laboratory of Physical and Structural Biology, NICHD/National Institutes of Health, 9 Memorial Drive, Bethesda, MD 20892-0924, USA.
J Biol Chem. 2006 Jun 16;281(24):16230-7. doi: 10.1074/jbc.M600650200. Epub 2006 Apr 14.
Using ion channel reconstitution in planar lipid bilayers, we examined the channel-forming activity of subfractions of Pseudomonas aeruginosa OprF, which was shown to exist in two different conformations: a minority single domain conformer and a majority two-domain conformer (Sugawara, E., Nestorovich, E. M., Bezrukov, S. M., and Nikaido, H. (2006) J. Biol. Chem. 281, 16220-16229). With the fraction depleted for the single domain conformer, we were unable to detect formation of any channels with well defined conductance levels. With the unfractionated OprF, we saw only rare channel formation. However, with the single domain-enriched fraction of OprF, we observed regular insertion of channels with highly reproducible conductances. Single OprF channels demonstrate rich kinetic behavior exhibiting spontaneous transitions between several subconformations that differ in ionic conductance and radius measured in polymer exclusion experiments. Although we showed that the effective radius of the most conductive conformation exceeds that of the general outer membrane porin of Escherichia coli, OmpF, we also found that a single OprF channel mainly exists in weakly conductive subconformations and switches to the fully open state for a short time only. Therefore, the low permeability of OprF reported earlier may be due to two factors: mainly to the paucity of the single domain conformer in the OprF population and secondly to the predominance of weakly conductive subconformations within the single domain conformer.
利用平面脂质双分子层中的离子通道重组技术，我们检测了铜绿假单胞菌OprF亚组分的通道形成活性，该蛋白存在两种不同构象：少数的单结构域构象体和多数的双结构域构象体（Sugawara, E., Nestorovich, E. M., Bezrukov, S. M., and Nikaido, H. (2006) J. Biol. Chem. 281, 16220 - 16229）。对于去除了单结构域构象体的组分，我们无法检测到具有明确电导水平的任何通道形成。对于未分级的OprF，我们仅观察到罕见的通道形成。然而，对于富含单结构域的OprF组分，我们观察到具有高度可重复电导的通道有规律地插入。单个OprF通道表现出丰富的动力学行为，在聚合物排阻实验中测量的离子电导和半径不同的几种亚构象之间呈现出自发转变。尽管我们表明最导电构象的有效半径超过了大肠杆菌的一般外膜孔蛋白OmpF，但我们也发现单个OprF通道主要以弱导电亚构象存在，仅在短时间内切换到完全开放状态。因此, 先前报道的OprF的低通透性可能归因于两个因素：主要是OprF群体中单结构域构象体的缺乏，其次是单结构域构象体内弱导电亚构象的占主导地位。
