Multivalent carbohydrate-lectin interactions are a key concept in biological processes mediating, e.g., signaling and adhesion. Binding affinities of multivalent ligands often increase by orders of magnitude compared to a monovalent binding situation. Thus, the design of multivalent ligands as potent inhibitors is a highly active field of research, where knowledge about the binding site topology is crucial. Here, we report a general strategy for precise distance measurements between the binding

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... sites of multivalent target proteins using monovalent ligands. We designed and synthesized Monovalent, conformationally Unambiguously Spin-labeled LIgands (MUeSLI). Inter-binding site distances of the complex model lectin wheat germ agglutinin were determined using pulsed electron paramagnetic resonance spectroscopy. This approach is an efficient way for exploring multivalent systems with monovalent ligands and it is readily transferable to other target proteins allowing the targeted design of multivalent ligands without structural information available. TOC GRAPHICS KEYWORDS EPR spectroscopy -DEER -distance measurements -spin labeling -multivalency -ligand design