The complex photophysics of 2-(2′-hydroxyphenyl)benzoxazole (HBO), 2-(2′-hydroxyphenyl)benzimidazole (HBI), and 2-(2′-hydroxyphenyl) benzothiazole (HBT) in different media makes them suitable as fluorescent probes to study the nature of binding sites in macromolecular systems. In this work, we investigate the spectroscopy of the three benzazole derivatives (HBXs) in different solvents and in human serum albumin (HSA) in order to understand the binding mechanism in subdomain IIA of HSA which has the ability to host a large variety of natural and pharmaceutical compounds. The three probes are found to specifically bind close to W214, the sole tryptophan residue in HSA, in a mode similar to that of the binding of the anticoagulant drug warfarin. The current results show that the structural differences between the three HBX molecules did not produce any measurable effects when binding with HSA. In particular, the change in planarity of the molecular backbone, from a perfectly planar and more rigid structure (HBO) to a twisted structure (HBI) and a flexible structure (HBT) has no effect on the mode of binding. Also, the strength of the intramolecular hydrogen bonds in HBXs (HBO > HBT > HBI) is shown not to intervene with the ability of HSA to ionize the ligands via through-space interaction with polar amino acid residues, similar to enzymatic reactions. The results emphasize the nature of HSA as a versatile and indiscriminate receptor, capable of binding a variety of ligands by adapting its binding pockets. In this regard, binding of HBXs, and other structurally similar ligands, in subdomain IIA is best described by the induced-fit model in which considerable flexibility of the binding site is necessary for molecular recognition. The results also point to the high affinity of the warfarin binding pocket (within subdomain IIA) for binding versatile molecular structures including several drugs. This affinity stems from the flexibility of the amino acids forming the binding pocket.
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