| 摘要 |
The present invention provides a method for determining the binding ability of a G-protein coupled receptor, hereinafter referred to as GPCR, and a mutateable ligand, said method comprising the steps of: a) providing a well microtiter plate having the wells disposed in a array having a first number of rows and a second number of columns; b) providing a GPCR, such as rhodopsin, in each of said wells; c) providing a number of mutants of the parent ligand, wherein the parent ligand being one that binds to the GPCR when the GPCR is residing in a particular conformation; d) bringing the mutants of the parent ligand in the wells into contact with the GPCR under conditions where the parent ligand would couple to the GPCR; and e) determining for each mutant whether the mutant ligand has a weaker or stronger binding ability as compared to the standard binding ability of the parent ligand and the GPCR by determining the amount of coupled mutant-GPCR complex in said wells. In an assay, rhodopsin binding of 403 mutants covering the complete arrestin sequence has been investigated. This information provides a functional 4th dimension to the crystal structures of inactive, preactivated and active arrestins. The resulting single amino acid resolution functional maps reveal a series of critical interactions in the polar core and along the C-tail of arrestin that are interrupted during arrestin activation. Our data further reveals several patches of amino acids that strongly reduce binding and act as direct binding interfaces to rhodopsin. This information in combination with computational molecular docking of active arrestin4 and light-activated rhodopsin allows to develop a model of the arrestin-rhodopsin complex. Combination of mutants allows modification of binding affinity and stability of the GPCR-ligand complex for diagnostic purposes or pharmacological intervention. |
