Evaluation by fluorescence, STD-NMR, docking and semi-empirical calculations of the o-NBA photo-acid interaction with BSA

Abstract Serum albumins present reversible pH dependent conformational transitions. A sudden laser induced pH-jump is a methodology that can provide new insights on localized protein (un)folding processes that occur within the nanosecond to microsecond time scale. To generate the fast pH jump needed to fast-trigger a protein conformational event, a photo-triggered acid generator as o -nitrobenzaldehyde ( o -NBA) can be conveniently used. In order to detect potential specific or nonspecific interactions between o -NBA and BSA, we have performed ligand-binding studies using fluorescence spectroscopy, saturation transfer difference (STD) NMR, molecular docking and semi-empirical calculations. Fluorescence quenching indicates the formation of a non-fluorescent complex in the ground-state between the fluorophore and the quencher, but o -NBA does not bind much effectively to the protein (K a  ~ 4.34 × 10 3  M − 1 ) and thus can be considered a relatively weak binder. The corresponding thermodynamic parameters: ΔG°, ΔS° and ΔH° showed that the binding process is spontaneous and entropy driven. Results of 1 H STD-NMR confirm that the photo-acid and BSA interact, and the relative intensities of the signals in the STD spectra show that all o -NBA protons are equally involved in the binding process, which should correspond to a nonspecific interaction. Molecular docking and semi-empirical calculations suggest that the o -NBA binds preferentially to the Trp-212-containing site of BSA (FA7), interacting via hydrogen bonds with Arg-217 and Tyr-149 residues.