Human serum albumin (HSA) is a key transport protein whose ability to bind multiple endogenous and exogenous ligands is governed by site heterogeneity and long-range conformational coupling; however, the mechanisms underlying ligand redistribution among binding sites, particularly in nanoparticulate HSA, remain poorly understood. To address this, we systematically examined the binding of ANS, DAUDA, palmitic acid (PA), and the anticancer lipopeptide PA-EQRPR to monomeric HSA (mHSA) and HSA nanoparticles (HSA-NPs) using steady-state and time-resolved fluorescence spectroscopy complemented by molecular modeling. In mHSA, three spectroscopically distinct binding species were resolved, with fluorescence lifetimes of ∼22.7, 14.5, and 1.6 ns and dissociation constants of 0.33, 9.0, and 3.3 μM, revealing multiple binding environments with distinct affinities and dynamics. Competitive binding experiments demonstrated cooperative PA binding and showed that PA-EQRPR not only displaces ANS or DAUDA but also promotes their redistribution to alternative, more hydrophobic sites, consistent with ligand-induced allosteric site-site communication. Lifetime-resolved analysis of DAUDA further revealed that PA stabilizes long-lived, high-affinity binding states, while PA-EQRPR shifts ligand populations toward deeper hydrophobic environments, enhancing fluorescence. HSA-NPs prepared using ethanol or acetone exhibited markedly different binding behaviors from mHSA, highlighting the impact of protein organization on ligand accessibility. Ethanol-induced HSA-NPs favored long-lifetime, hydrophobic binding species, whereas acetone-induced particles showed reduced site heterogeneity. Docking and molecular dynamics simulations revealed ligand-driven conformational rearrangements that reshape HSA's hydrophobicity landscape. Together, these findings introduce an allosteric population-shift framework that rationalizes multisite ligand binding and redistribution in both monomeric and nanoparticulate HSA.
(Guliyeva) et al. (Sat,) studied this question.