Mayer Michael, Semetey Vincent, Gitlin Irina, Yang Jerry, Whitesides George M
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
J Am Chem Soc. 2008 Jan 30;130(4):1453-65. doi: 10.1021/ja077555f. Epub 2008 Jan 8.
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 approximately 30 kD) to the ion channel-forming peptides (MW approximately 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 approximately 2 microM 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.
本文提出了一种通过触发平面脂质双层中离子通道形成肽的自组装破坏来传感亲和相互作用的方法。研究表明，携带共价连接的磺酰胺配体的短杆菌肽衍生物与碳酸酐酶II（CA II）的结合导致通过双层的离子通道电导受到抑制。我们认为，体积较大的CA II蛋白（分子量约30 kD）与离子通道形成肽（分子量约2.5 kD）的结合，要么降低了这些肽自组装成孔的倾向，要么将它们完全从双层中提取出来。在这两种情况下，蛋白质与配体之间的相互作用都会导致自组装孔的破坏。向溶液中添加竞争性抑制剂4-羧基苯磺酰胺，可使CA II从短杆菌肽-磺酰胺缀合物中释放出来，并通过允许双层中的离子通道重新组装来恢复跨双层的电流流动。在离散时间间隔内对电流进行时间平均记录，使得量化这种单价配体结合相互作用成为可能。该方法得出在双层记录室中CA II与短杆菌肽-磺酰胺结合的解离常数约为2 microM：该值与通过等温滴定量热法独立获得的CA II与相关磺酰胺衍生物的值一致。
