Using ion channel-forming peptides to quantify protein-ligand interactions.
Using ion channel-forming peptides to quantify protein-ligand interactions.
复制标题
使用离子通道形成肽来量化蛋白质-配体相互作用。
DOI：
10.1021/ja077555f
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发表时间：
2008
影响因子：
15
通讯作者：
Whitesides,GeorgeM
中科院分区：
文献类型：
--
作者：
Mayer,Michael;Semetey,Vincent;Gitlin,Irina;Yang,Jerry;Whitesides,GeorgeM
本文提出了一种通过触发破坏平面脂质双层中离子通道形成肽的自组装来检测亲和相互作用的方法。它表明，丙甲霉素的衍生物携带共价连接的磺酰胺配体的碳酸酐酶II（CA II）的结合导致通过双层的离子通道电导的抑制。我们提出，大体积的CA II蛋白（分子量约30 kD）的离子通道形成肽（分子量约2.5 kD）的结合，要么降低了这些肽的趋势，自组装成一个孔或提取它们从双层完全。在这两种结果中，蛋白质和配体之间的相互作用都会导致自组装孔的破坏。添加竞争性抑制剂，4-羧基苯磺酰胺，从丙氨甲素-磺酰胺共轭物的溶液中释放CA II，并通过允许在双层中的离子通道的重新组装来恢复穿过双层的电流。在离散的时间间隔内的电流的时间平均记录使得有可能量化这种单价配体结合相互作用。该方法在双层记录室中给出了CA II与丙氨酰青霉素-磺酰胺结合的解离常数为1.2 μM：该值与通过等温滴定量热法独立获得的CA II和相关磺酰胺衍生物的值一致。
This paper proposes a method for sensing affinity interactions by triggering disruption of self-assembly of ion channel-forming peptides in planar lipid bilayers. It shows that the binding of a derivative of alamethicin carrying a covalently attached sulfonamide ligand to carbonic anhydrase II (CA II) resulted in the inhibition of ion channel conductance through the bilayer. We propose that the binding of the bulky CA II protein (MW ≈ 30 kD) to the ion channel-forming peptides (MW ≈ 2.5 kD) either reduced the tendency of these peptides to self-assemble into a pore or extracted them from the bilayer altogether. In both outcomes, the interactions between the protein and the ligand lead to a disruption of self-assembled pores. Addition of a competitive inhibitor, 4-carboxybenzenesulfonamide, to the solution released CA II from the alamethicin-sulfonamide conjugate and restored the current flow across the bilayer by allowing reassembly of the ion channels in the bilayer. Time-averaged recordings of the current over discrete time intervals made it possible to quantify this monovalent ligand binding interaction. This method gave a dissociation constant of ∼2 μM for the binding of CA II to alamethicin-sulfonamide in the bilayer recording chamber: this value is consistent with a value obtained independently with CA II and a related sulfonamide derivative by isothermal titration calorimetry.