Designed Anchoring Geometries Determine Lifetimes of Biotin-Streptavidin Bonds under Constant Load and Enable Ultra-Stable Coupling

Abstract

The small molecule biotin and the homotetrameric protein streptavidin (SA) form a stable and robust complex that plays a pivotal role in many biotechnological and medical applications. In particular, the biotin-streptavidin linkage is frequently used in single molecule force spectroscopy (SMFS) experiments. Recent data suggest that biotin-streptavidin bonds show strong directional dependence and a broad range of multi-exponential lifetimes under load. Here, we investigate engineered SA variants with different valencies and a unique tethering point under constant forces using a magnetic tweezer assay. We observed two orders-of-magnitude differences in the lifetimes, which we attribute to the distinct force loading geometries in the different SA variants. We identified an especially long-lived tethering geometry that will facilitate ultra-stable SMFS experiments and pave the way for new biotechnological applications.