RESUMO
Abscisic acid (ABA, 1) is a plant hormone that regulates various plant physiological processes such as seed developing and stress responses. The ABA signaling system has been elucidated; binding of ABA with PYL proteins triggers ABA signaling. We have previously reported a new method to isolate a protein targeted with a bioactive small molecule using a biotin linker with alkyne and amino groups, a protein cross-linker, and a bioactive small molecule with an azido group (azido probe). This method was used to identify the unknown ABA binding protein of Arabidopsis thaliana. As a result, AtTrxh3, a thioredoxin, was isolated as an ABA binding protein. Our developed method can be applied to the identification of binding proteins of bioactive compounds.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Tiorredoxinas/metabolismo , Ácido Abscísico/química , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Transporte , Cromatografia Líquida , Estrutura Molecular , Ligação Proteica , Proteoma , Proteômica/métodos , Espectrometria de Massas em Tandem , Tiorredoxinas/química , Tiorredoxinas/genética , Tiorredoxinas/isolamento & purificaçãoRESUMO
Target protein identification of bioactive small molecules is one of the most important research in forward chemical genetics. The affinity chromatography technique to use a resin bound with a small molecule is often used for identification of a target protein of a bioactive small molecule. Here we report a new method to isolate a protein targeted with a bioactive small molecule using a biotin linker with alkyne and amino groups, protein cross-linker containing disulfide bond, and a bioactive small molecule with an azido group (azido probe). After an azido probe is associated with a target protein, the complex of a target protein and azido probe is covalently bound through the biotin linker by azide-alkyne Huisgen cycloaddition and protein cross-linker containing disulfide bond. This ternary complex is immobilized on an affinity matrix with streptavidin, and then the target protein is selectively eluted with a buffer containing a reducing agent for cleavage of disulfide bonds. This method uses a probe having an azido group, which a small functional group, and has the possibility to be a solution strategy to overcome the hindrance of a functional group introduced into the probe that reduces association a target protein. The effectiveness of the method in this study was shown using linker 1, 3'-azidoabscisic acid 3, and protein cross-linker containing a disulfide bond (DTSSP 5).
Assuntos
Ácido Abscísico/metabolismo , Alcinos/química , Aminas/química , Biotina/química , Proteínas de Plantas/química , Proteínas Recombinantes/química , Estreptavidina/química , Ácido Abscísico/análogos & derivados , Ácido Abscísico/química , Proteínas de Arabidopsis/genética , Azidas/química , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Reagentes de Ligações Cruzadas/química , Reação de Cicloadição , Dissulfetos/química , Escherichia coli/química , Escherichia coli/genética , Oxirredução , Proteínas de Plantas/genética , Proteínas Recombinantes/genética , Succinimidas/química , Espectrometria de Massas em TandemRESUMO
We synthesized a novel linker (1) with biotin, alkyne and amino groups for the identification of target proteins using a small molecule that contains an azide group (azide probe). The alkyne in the linker bound the azide probe via an azide-alkyne Huisgen cycloaddition. A protein cross-linker effectively bound the conjugate of the linker and an azide probe with a target protein. The covalently bound complex was detected by western blotting. Linker 1 was applied to a model system using an abscisic acid receptor, RCAR/PYR/PYL (PYL). Cross-linked complexes of linker 1, the azide probes and the target proteins were successfully visualized by western blotting. This method of target protein identification was more effective than a previously developed method that uses a second linker with biotin, alkyne, and benzophenone (linker 2) that acts to photo-crosslink target proteins. The system developed in this study is a method for identifying the target proteins of small bioactive molecules and is different from photo-affinity labelling.
Assuntos
Alcinos/química , Proteínas de Arabidopsis/química , Biotina/análogos & derivados , Biotina/química , Sondas Moleculares/química , Alcinos/síntese química , Arabidopsis/química , Azidas/síntese química , Azidas/química , Biotina/síntese química , Western Blotting , Química Click , Reagentes de Ligações Cruzadas/síntese química , Reagentes de Ligações Cruzadas/química , Reação de Cicloadição , Escherichia coli/química , Proteínas de Escherichia coli/química , Oxirredutases Intramoleculares/química , Luminescência , Lisina/análogos & derivados , Lisina/síntese química , Lisina/química , Sondas Moleculares/síntese químicaRESUMO
A novel linker containing biotin, alkyne and benzophenone groups (1) was synthesized to identify target proteins using a small molecule probe. This small molecule probe contains an azide group (azide probe) that reacts with an alkyne in 1 via an azide-alkyne Huisgen cycloaddition. Cross-linking of benzophenone to the target protein formed a covalently bound complex consisting of the azide probe and the target protein via 1. The biotin was utilized via biotin-avidin binding to identify the cross-linked complex. To evaluate the effectiveness of 1, it was applied in a model system using an allene oxide synthase (AOS) from the model moss Physcomitrella patens (PpAOS1) and an AOS inhibitor that contained azide group (3). The cross-linked complex consisting of PpAOS1, 1 and 3 was resolved via SDS-PAGE and visualized using a chemiluminescent system. The method that was developed in this study enables the effective identification of target proteins.