Kleivdal H, Benz R, Jensen H B
Department of Biochemistry and Molecular Biology, University of Bergen, Norway.
Eur J Biochem. 1995 Oct 1;233(1):310-6. doi: 10.1111/j.1432-1033.1995.310_1.x.
The pore-forming activity of the major outer-membrane protein FomA of the anaerobic Fusobacterium nucleatum was studied in artificial lipid bilayer membranes. FomA was isolated from F. nucleatum strains Fev1, ATCC 10953, and ATCC 25586 by extraction with lithium dodecyl sulfate and lithium chloride and had an apparent molecular mass of about 40 kDa. When solubilized at low temperatures, the protein ran with an apparent molecular mass of about 62 kDa on SDS/PAGE. Cross-linking experiments and two-dimensional SDS/PAGE gave evidence that the 62-kDa protein band represented the trimeric form of FomA. The protein trimers were susceptible to SDS and temperature. The stability of the porin trimers varied among the strains. The properties of the FomA channels were studied in reconstitution experiments with black lipid bilayer membranes. The F. nucleatum porins formed channels with single-channel conductances in the range 0.66-1.30 nS in M KCl. The single-channel conductance was a function of the mobilities of the ions present in the aqueous solution bathing the bilayer membrane. This means that FomA forms general diffusion channels since (a) the conductance showed a linear dependence on the salt concentration, (b) the ion selectivity was small and varied for the three strains, and (c) the channels did not exhibit any binding site for maltotriose or triglycine. The water-filled channel was voltage dependent, and conductance decrements were observed at transmembrane potentials of +/- 50 mV. The conductance decrement steps were about one-third of the total conductance of a functional unit in its fully 'open' state. This strongly suggests that the trimer is the functional unit of the porin.
在人工脂质双层膜中研究了具核梭杆菌主要外膜蛋白FomA的成孔活性。通过用十二烷基硫酸锂和氯化锂提取，从具核梭杆菌菌株Fev1、ATCC 10953和ATCC 25586中分离出FomA，其表观分子量约为40 kDa。当在低温下溶解时，该蛋白在SDS/PAGE上的表观分子量约为62 kDa。交联实验和二维SDS/PAGE表明，62 kDa的蛋白条带代表FomA的三聚体形式。蛋白三聚体对SDS和温度敏感。孔蛋白三聚体的稳定性在不同菌株间有所不同。在黑脂质双层膜的重构实验中研究了FomA通道的特性。具核梭杆菌孔蛋白在1 M KCl中形成单通道电导范围为0.66 - 1.30 nS的通道。单通道电导是双层膜外水浴溶液中离子迁移率的函数。这意味着FomA形成一般扩散通道，因为：(a) 电导对盐浓度呈线性依赖；(b) 离子选择性小且三种菌株有所不同；(c) 通道对麦芽三糖或三甘氨酸没有任何结合位点。充满水的通道依赖电压，在±50 mV的跨膜电位下观察到电导下降。电导下降步骤约为功能单元在完全“开放”状态下总电导的三分之一。这强烈表明三聚体是孔蛋白的功能单元。
