Identification of interaction partners using protein aggregation and NMR spectroscopy.

The interaction among proteins is one of the most fundamental methods of information transfer in the living system. Many methods have been developed in order to identify the interaction pairs or groups either in vivo or in vitro. The in vitro pulldown/coprecipitation assay directly observes the protein that binds to the target. This method involves electrophoresis, which is a technique of a low resolution as well as a low throughput. As a better alternative, we wish to propose a new method that is based on the NMR spectroscopy. This method utilizes the aggregation of the target protein and the concomitant signal disappearance of the interacting partner. The aggregation is accomplished by the elastin-like polypeptide, which is fused to the target. If a protein binds to this supramolecular complex, its NMR signal then becomes too broadened in order to be observed, which is the basic phenomenon of the NMR spectroscopy. Thus, the protein that loses its signal is the one that binds to the target. A compound that interferes with these types of bindings among the proteins can be identified by observing the reappearance of the protein signals with the simultaneous disappearance of the signals of the compound. This technique will be applied in order to find an interaction pair in the information transfer pathway as well as a compound that disrupts it. This proposed method should be able to work with a mixture of proteins and provide a higher resolution in order to find the binding partner in a higher throughput fashion.