Liu Jing, Strzalka Joseph, Tronin Andrey, Johansson Jonas S, Blasie J Kent
Departments of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Biophys J. 2009 May 20;96(10):4176-87. doi: 10.1016/j.bpj.2009.01.055.
We demonstrate that cyano-phenylalanine (Phe(CN)) can be utilized to probe the binding of the inhalational anesthetic halothane to an anesthetic-binding, model ion channel protein hbAP-Phe(CN). The Trp to Phe(CN) mutation alters neither the alpha-helical conformation nor the 4-helix bundle structure. The halothane binding properties of this Phe(CN) mutant hbAP-Phe(CN), based on fluorescence quenching, are consistent with those of the prototype, hbAP1. The dependence of fluorescence lifetime as a function of halothane concentration implies that the diffusion of halothane in the nonpolar core of the protein bundle is one-dimensional. As a consequence, at low halothane concentrations, the quenching of the fluorescence is dynamic, whereas at high concentrations the quenching becomes static. The 4-helix bundle structure present in aqueous detergent solution and at the air-water interface, is preserved in multilayer films of hbAP-Phe(CN), enabling vibrational spectroscopy of both the protein and its nitrile label (-CN). The nitrile groups' stretching vibration band shifts to higher frequency in the presence of halothane, and this blue-shift is largely reversible. Due to the complexity of this amphiphilic 4-helix bundle model membrane protein, where four Phe(CN) probes are present adjacent to the designed cavity forming the binding site within each bundle, all contributing to the infrared absorption, molecular dynamics (MD) simulation is required to interpret the infrared results. The MD simulations indicate that the blue-shift of -CN stretching vibration induced by halothane arises from an indirect effect, namely an induced change in the electrostatic protein environment averaged over the four probe oscillators, rather than a direct interaction with the oscillators. hbAP-Phe(CN) therefore provides a successful template for extending these investigations of the interactions of halothane with the model membrane protein via vibrational spectroscopy, using cyano-alanine residues to form the anesthetic binding cavity.
我们证明，氰基苯丙氨酸（Phe(CN)）可用于探测吸入性麻醉剂氟烷与一种麻醉剂结合的模型离子通道蛋白hbAP-Phe(CN)的结合情况。色氨酸突变为Phe(CN)既不改变α-螺旋构象，也不改变四螺旋束结构。基于荧光猝灭，这种Phe(CN)突变体hbAP-Phe(CN)的氟烷结合特性与原型hbAP1一致。荧光寿命随氟烷浓度的变化表明，氟烷在蛋白束非极性核心中的扩散是一维的。因此，在低氟烷浓度下，荧光猝灭是动态的，而在高浓度下，猝灭变为静态。存在于水性洗涤剂溶液和空气-水界面的四螺旋束结构，在hbAP-Phe(CN)的多层膜中得以保留，从而能够对蛋白质及其腈标记物（-CN）进行振动光谱分析。在氟烷存在下，腈基的伸缩振动带向高频移动，并且这种蓝移在很大程度上是可逆的。由于这种两亲性四螺旋束模型膜蛋白的复杂性，其中四个Phe(CN)探针存在于每个束内形成结合位点的设计腔附近，所有这些都对红外吸收有贡献，因此需要分子动力学（MD）模拟来解释红外结果。MD模拟表明，氟烷引起的-CN伸缩振动的蓝移源于间接效应，即四个探针振荡器平均的蛋白质静电环境的诱导变化，而不是与振荡器的直接相互作用。因此，hbAP-Phe(CN)为通过振动光谱扩展对氟烷与模型膜蛋白相互作用的这些研究提供了一个成功的模板，利用氰基丙氨酸残基形成麻醉剂结合腔。
