Fluorescence spectroscopy as a tool for investigating molecular interactions with proteins

Fluorescence spectroscopy has proven to be a valuable tool for characterizing interactions between small molecules and proteins. In this talk, the key considerations for proper data processing will be discussed, with particular emphasis on the correction of the inner filter effect, the neglect of which can lead to spectral distortions, resulting in red/blue shifts and misleading interpretations. The mechanisms of fluorescence quenching will also be discussed, as well as how to use theoretical models applied to fluorescence data to characterize the binding constant and extract thermodynamic parameters of the interaction, such as Gibbs free energy, enthalpy, and entropy. The use of the phenomenon of Fluorescence Resonance Energy Transfer (FRET) as a spectroscopic ruler for protein–ligand systems will be explored. Particular attention will be given to the importance of correcting the data based on binding equilibrium, as well as to the influence of ligand stabilization within the binding site, which directly affects the calculation of the dipole orientation factor (κ²). Finally, the talk will discuss how molecular dynamics simulations can complement these models, providing structural and dynamic support to the experimental analysis.