RH421 is a voltage-sensitive fluorescent styrylpyridinium dye which has often been used to probe the kinetics of Na+,K+-ATPase partial reactions. The origin of the dye's response has up to now been unclear. Here we show that RH421 responds to phosphorylation of the Na+,K+-ATPase by inorganic phosphate with a fluorescence increase. Analysis of the kinetics of the fluorescence response indicates that the probe is not detecting phosphorylation itself but rather a shift in the protein's E1/E2 conformational equilibrium induced by preferential phosphate binding to and phosphorylation of enzyme in the E2 conformation. Molecular dynamics simulations of crystal structures in lipid bilayers indicate some change in the protein's hydrophobic thickness during the E1-E2 transition, which may influence the dye response. However, the transition is known to involve significant rearrangement of the protein's highly charged lysine-rich cytoplasmic N-terminal sequence. Using poly-l-lysine as a model of the N-terminus, we show that an analogous response of RH421 to the E1→E2P conformational change is produced by poly-l-lysine binding to the surface of the Na+,K+-ATPase-containing membrane fragments. Thus, it seems that the prime origin of the RH421 fluorescence response is a change in the interaction of the protein's N-terminus with the surrounding membrane. Quantum mechanical calculations of the dye's visible absorption spectrum give further support to this conclusion. The results obtained indicate that membrane binding and release of the N-terminus of the Na+,K+-ATPase α-subunit are intimately involved in the protein's catalytic cycle and could represent an effective site of regulation.

中文翻译：

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压敏染料RH421检测膜表面的Na +，K + -ATPase构象变化。

RH421是一种电压敏感型荧光苯乙烯基吡啶鎓染料，通常用于探测Na +，K + -ATPase部分反应的动力学。到目前为止，染料反应的起源还不清楚。在这里，我们显示RH421通过无机磷酸盐响应Na +，K + -ATPase的磷酸化，并具有荧光增强作用。荧光反应动力学的分析表明，该探针本身未检测到磷酸化，而是检测了蛋白质的E1 / E2构象平衡的偏移，该平衡是由优先磷酸结合到E2构象中的酶和磷酸化引起的。脂质双层中晶体结构的分子动力学模拟表明，在E1-E2过渡过程中蛋白质的疏水厚度发生了一些变化，这可能会影响染料的响应。然而，已知该转变涉及该蛋白质的高度带电荷的富含赖氨酸的胞质N-末端序列的显着重排。使用聚-1-赖氨酸作为N端的模型，我们表明RH421对E1→E2P构象变化的类似响应是通过聚1-赖氨酸结合到Na +，K + -ATPase-的表面而产生的。含有膜碎片。因此，似乎RH421荧光反应的主要来源是蛋白质N末端与周围膜相互作用的变化。染料可见吸收光谱的量子力学计算为这一结论提供了进一步的支持。获得的结果表明，膜结合和Na +，K + -ATPaseα-亚基的N-末端的释放与蛋白质'密切相关。