Probing the Hydrogen Bond Involving Acridone Trapped in a Hydrophobic Biological Nanocavity: Integrated Spectroscopic...

Spectroscopic analyses reveal that acridone (AD) penetrates through the structure and enters the hydrophobic cavity of the protein β-lactoglobulin (βLG). Although the protein contains two tryptophan (Trp) residues, AD interacts with only one (Trp-19), which is authenticated by the appearance of a single isoemissive point in TRANES. Alteration in the secondary structure of the protein while AD pierces through βLG is evident from the circular dichroism spectroscopic study. The ground-state interaction between AD and βLG is proven from the UV-vis spectroscopic study and the static nature of quenching of intrinsic fluorescence of the protein by the ligand. The steady-state fluorescence study in varied temperatures indicates the involvement of hydrogen bonding in the ligand-protein interaction. Further, the time-resolved fluorescence anisotropy study gives a hint of the presence of a hydrogen bond in AD-βLG interaction, which possibly involves the rotamers of Trp-19. In fact, the idea of involvement of rotamers of Trp-19 is obtained from the increase in fluorescence lifetime of βLG in the presence of AD. The docking study agrees to the involvement of hydrogen bonding in AD-βLG interaction. The direct evidence of hydrogen bonding between Trp and AD is obtained from the laser flash photolysis studies where the signature of formation of ADH^• and Trp^• through hydrogen abstraction between Trp and AD, loosely bound through hydrogen bonding, gets prominence. Thus, binding of AD to βLG involves hydrogen bonding in a hydrophobic pocket of the protein.