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The complex of the small molecule biotin and the homotetrameric protein streptavidin is key to a broad range of biotechnological applications. Therefore, the behavior of this extraordinarily high-affinity interaction under mechanical force is intensively studied by single-molecule force spectroscopy. Recently, steered molecular dynamics simulations have identified a low force pathway for the dissociation of biotin from streptavidin, which involves partial unfolding of the N-terminal β-sheet structure of monovalent streptavidin's functiona