ABSTRACT
We demonstrate a novel NMR method for the mapping of protein-protein interaction sites. In our approach protein-protein binding sites are mapped by competition binding experiments using indirect NMR reporter technology and Ala positional scanning. The methodology provides high sensitivity, ease of implementation and high-throughput capabilities. The feasibility of the technique is demonstrated with an application to the beta-Catenin/Tcf4 complex.
Subject(s)
Mutation/genetics , Nuclear Magnetic Resonance, Biomolecular/methods , Protein Interaction Mapping/methods , Binding, Competitive , Humans , Ligands , Models, Molecular , Reproducibility of Results , TCF Transcription Factors/metabolism , Transcription Factor 7-Like 2 Protein , beta Catenin/metabolismABSTRACT
A novel method is proposed for the detection and quantification of protein-protein interactions in solution. In this approach, one protein binding partner is tagged with a ligand binding domain, and protein-protein interaction is monitored via changes in the NMR relaxation of a reporter ligand which reversibly binds to the ligand binding domain. The particular benefit of the method is that only minute amounts of protein material and no isotope labeling are required. Its ease of implementation and the high-throughput capabilities make the method an attractive complement to existing proteomic methodology.
