Binding of Biotin to Streptavidin: A combined fluorescence correlation spectroscopy and time-resolved fluorescence study

The Biotin-Streptavidin complex is a widely studied system in biology and biophysics, because of its extremely strong non-covalent binding affinity. The latter is often exploited to link molecules to substrates or to one another. However, the details of the Biotin-Streptavidin binding have not been fully elucidated so far. Particularly, the role of cooperative effects in enhancing the binding affinity has not been clarified. Our long-term aim is to investigate this point by implementing two complementary approaches, fluorescence correlation spectroscopy and time-correlated single-photon counting. As both methods rely on the analysis of fluorescence signals, biotin labeled with Atto-550-dye was used. In this work, in order to get a first overview of the system, we analyzed solutions in three paradigmatic ranges of Biotin-to-Streptavidin concentration ratio. Fluorescence correlation spectroscopy measurements allowed us to extract diffusion times of free biotin and of biotin-Streptavidin complexes, and also to gain information about the dynamics of the intersystem crossing between the first excited triplet and the first excited singlet states. Time-correlated single-photon counting made it possible to derive the lifetimes of the different species in solution, as well as to deduce relevant information about the relative abundance of Streptavidin-complexed and free Biotin.