Mapping proteinâ??protein interactions by double-REDOR-filtered magic angle spinning NMR spectroscopy

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Mapping proteinâ??protein interactions by double-REDOR-filtered magic angle spinning NMR spectroscopy

Abstract

REDOR-based experiments with simultaneous 1Hâ??13C and 1Hâ??15N dipolar dephasing are explored for investigating intermolecular proteinâ??protein interfaces in complexes formed by a Uâ??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 Uâ??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 1Hâ??13C or 1Hâ??15N cross polarization, permitting to establish the residues of the Uâ??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 13Câ??13C correlation experiments. We established the validity of this approach on Uâ??13C,15N-histidine and on a structurally characterized complex of dynactinâ??s Uâ??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.



Source: Journal of Biomolecular NMR