Changmiao Guo1, Guangjin Hou2, Xingyu Lu1, Tatyana Polenova3. 1. Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, 19716, USA. 2. Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, 19716, USA. hou@udel.edu. 3. Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, 19716, USA. tpolenov@udel.edu.
Abstract
REDOR-based experiments with simultaneous 1H-13C and 1H-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 by 1H-13C or 1H-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 into 13C-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.
REDOR-based experiments with simultaneous 1H-13C and n class="Chemical">1H-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 by 1H-13C or 1H-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 into 13C-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.
Entities:
Keywords:
Double REDOR; Magic angle spinning; Protein interfaces
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