Hancock R E, Benz R
Biochim Biophys Acta. 1986 Sep 11;860(3):699-707. doi: 10.1016/0005-2736(86)90569-9.
The interaction of phosphate ions with the Pseudomonas aeruginosa anion-specific protein P channel was probed. The single-channel conductance of protein P incorporated into planar lipid bilayer membranes in the presence of 0.3 M H2PO-4 was shown to be 6.0 pS, demonstrating that protein P channels allowed the permeation of phosphate. When large numbers of protein P channels were incorporated into lipid bilayer membranes in the presence of 40 mM Cl-, addition of small concentrations of phosphate resulted in reduction of macroscopic Cl- conductance in a dose- (and pH-) dependent fashion. This allowed calculation of an I50 value of e.g. 0.46 mM at pH 7.0, suggesting that the affinity of protein P for its normal substrate phosphate was at least 60-100-fold greater than the affinity of the channel for other ions such as chloride. Pyrophosphate and the phosphate analogue, arsenate, also inhibited macroscopic Cl- conductance through protein P with I50 values at pH 7.0 of 4.9 mM and 1.3 mM, respectively. To probe the nature of the phosphate binding site, the epsilon-amino groups of available lysine residues of protein P were chemically modified. Acetylation and carbamylation which produced uncharged, modified lysines destroyed both the anion (e.g. Cl-) binding site and the phosphate binding site as determined by single-channel experiments and macroscopic conductance inhibition experiments respectively. Nevertheless, the modified proteins still retained their trimeric configuration and their ability to reconstitute single channels in lipid bilayer membranes. Methylation, which allowed retention of the charge on the modified lysine residues, increased the Kd of the channel for Cl- 33-fold and the I50 for phosphate inhibition of macroscopic Cl- conductance 2.5-4-fold. A molecular model for the phosphate binding site of the protein P channel is presented.
对磷酸根离子与铜绿假单胞菌阴离子特异性蛋白P通道之间的相互作用进行了探究。结果表明，在存在0.3 M H2PO4-的情况下，整合到平面脂质双分子层膜中的蛋白P的单通道电导为6.0 pS，这表明蛋白P通道允许磷酸根渗透。当在存在40 mM Cl-的情况下将大量蛋白P通道整合到脂质双分子层膜中时，添加低浓度的磷酸盐会导致宏观Cl-电导以剂量（和pH）依赖性方式降低。由此计算出例如在pH 7.0时的I50值为0.46 mM，这表明蛋白P对其正常底物磷酸根的亲和力比对其他离子（如氯离子）的通道亲和力至少高60 - 100倍。焦磷酸和磷酸盐类似物砷酸盐也抑制通过蛋白P的宏观Cl-电导，在pH 7.0时的I50值分别为4.9 mM和1.3 mM。为了探究磷酸根结合位点的性质，对蛋白P中可用赖氨酸残基的ε-氨基进行了化学修饰。通过单通道实验和宏观电导抑制实验分别确定，产生不带电修饰赖氨酸的乙酰化和氨甲酰化破坏了阴离子（如Cl-）结合位点和磷酸根结合位点。然而，修饰后的蛋白质仍保留其三聚体结构以及在脂质双分子层膜中重构单通道的能力。允许修饰赖氨酸残基上电荷保留的甲基化使通道对Cl-的Kd增加了33倍，使宏观Cl-电导的磷酸盐抑制I50增加了2.5 - 4倍。本文提出了蛋白P通道磷酸根结合位点的分子模型。
