Single Molecule Studies of Streptavidin-Biotin Dissociation at Elevated Temperatures

Abstract

Over the past 10 years single molecule spectroscopic techniques have matured and are widely used in the biomolecular sciences. More recently, temperature control has been incorporated, increasing the dimensionality of the possible experimental parameters. However, conventional immobilization techniques rely on biotin-streptavidin linkages to localize single molecules on surfaces. While this ligand binding interaction is one of the strongest non-covalent linkages in nature (KD≈10-14 M) and is extremely stable at room temperature, the kinetics of dissociation at elevated temperatures are poorly understood. Pulsed laser based IR absorbance heating and fluorescence techniques are used to measure the dissociation rate at temperatures well above room temperature. The rate constants of dissociation are found to increase significantly from ∼2x10-5 s-1 at 20°C to ∼1x10-3 s-1 at 55°C. Eyring analysis reveals that the dissociation reaction has a large enthalpic contribution (ΔH‡ = 20 (1) kcal/mol), the magnitude of which is on the order of the previously measured enthalpies of formation. This result indicates that the biotin binding occurs over a nearly barrier-less transition state.