title: Mapping protein-protein interactions by double-REDOR-filtered magic angle spinning NMR spectroscopy.
abstract: REDOR-based experiments with simultaneous1H-13C and1H-15N dipolar dephasing are explored for investigating intermolecular protein-protein interfaces in complexes formed by a U-13C,15N-labeled protein and its natural abundance binding partner. The application of a double-REDOR filter (dREDOR) results in a complete dephasing of proton magnetization in the U-13C,15N-enriched molecule while the proton magnetization of the unlabeled binding partner is not dephased. This retained proton magnetization is then transferred across the intermolecular interface by1H-13C or1H-15N cross polarization, permitting to establish the residues of the U-13C,15N-labeled protein, which constitute the binding interface. To assign the interface residues, this dREDOR-CPMAS element is incorporated as a building block into13C-13C correlation experiments. We established the validity of this approach on U-13C,15N-histidine and on a structurally characterized complex of dynactin's U-13C,15N-CAP-Gly domain with end-binding protein 1 (EB1). The approach introduced here is broadly applicable to the analysis of intermolecular interfaces when one of the binding partners in a complex cannot be isotopically labeled.
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, 19716, USA.
authors.forename: Changmiao
authors.initials: C
authors.lastname: Guo
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, 19716, USA. hou@udel.edu.
authors.forename: Guangjin
authors.initials: G
authors.lastname: Hou
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, 19716, USA.
authors.forename: Xingyu
authors.initials: X
authors.lastname: Lu
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, 19716, USA. tpolenov@udel.edu.
authors.forename: Tatyana
authors.identifier: 0000-0002-0346-1131
authors.initials: T
authors.lastname: Polenova
mesh_terms: D003198:Computer Simulation; D006639:Histidine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D011506:Proteins / Q000737:chemistry*
chemical_list: D011506:Proteins; D006639:Histidine
keywords: Double REDOR; Magic angle spinning; Protein interfaces