Rokitskaya T I, Antonenko Y N, Kotova E A, Anastasiadis A, Separovic F
Belozersky Institute, Moscow State University, Moscow 119899, Russia.
Biochemistry. 2000 Oct 24;39(42):13053-8. doi: 10.1021/bi0007876.
Membrane protein functioning basically depends on the supramolecular structure of the proteins which can be modulated by specific interactions with external ligands. The effect of a water-soluble protein bearing specific binding sites on the kinetics of ionic channels formed by gramicidin A (gA) in planar bilayer lipid membranes (BLM) has been studied using three independent approaches: (1) sensitized photoinactivation, (2) single-channel, and (3) autocorrelation measurements of current fluctuations. As shown previously [Rokitskaya, T. I., et al. (1996) Biochim. Biophys. Acta 1275, 221], the time course of the flash-induced current decrease in most cases follows a single-exponential decay with an exponential factor (tau) that corresponds to the gA single-channel lifetime. Addition of avidin does not affect tau for gA channels, but causes a dramatic increase in tau for channels formed by gA5XB, a biotinylated analogue of gA. This effect is reversed by addition of an excess of biotin to the bathing solution. The average single-channel duration of gA5XB was about 3.6 s as revealed by single-channel recording of the BLM current. After prolonged incubation with avidin, a long-lasting open state of the gA5XB channel appeared which did not close for more than 10 min. The data on gA5XB photoinactivation kinetics and single-channel measurements were confirmed by analysis of the corresponding power spectra of the current fluctuations obtained in the control, in the presence of avidin, and after the addition of biotin. We infer that avidin produces a deceleration of gA5XB channel kinetics by motional restriction of gA5XB monomers and dimers upon the formation of avidin and gA5XB complexes, which would stabilize the channel state and thus increase the single-channel lifetime.
膜蛋白的功能基本上取决于蛋白质的超分子结构，这种结构可通过与外部配体的特异性相互作用进行调节。使用三种独立的方法研究了一种带有特异性结合位点的水溶性蛋白对平面双层脂质膜（BLM）中短杆菌肽A（gA）形成的离子通道动力学的影响：（1）敏化光灭活，（2）单通道，以及（3）电流波动的自相关测量。如先前所示[罗基茨卡娅，T. I.等人（1996年）《生物化学与生物物理学报》1275卷，221页]，在大多数情况下，闪光诱导电流下降的时间过程遵循单指数衰减，其指数因子（τ）对应于gA单通道寿命。添加抗生物素蛋白不会影响gA通道的τ，但会导致gA的生物素化类似物gA5XB形成的通道的τ显著增加。向浴液中添加过量生物素可逆转这种效应。通过对BLM电流进行单通道记录发现，gA5XB的平均单通道持续时间约为3.6秒。与抗生物素蛋白长时间孵育后，gA5XB通道出现了持续开放状态，持续时间超过10分钟。通过分析在对照、存在抗生物素蛋白以及添加生物素后获得的电流波动的相应功率谱，证实了关于gA5XB光灭活动力学和单通道测量的数据。我们推断，抗生物素蛋白通过在形成抗生物素蛋白与gA5XB复合物时对gA5XB单体和二聚体的运动限制，导致gA5XB通道动力学减速，这将稳定通道状态，从而增加单通道寿命。
