Conformational constraints and ligand interactions are key determinants of the distinct aggregation pathways observed in human serum albumin.

Protein aggregation is a fundamental phenomenon linked to many amyloid-associated disorders, production of therapeutic proteins and biomaterial. Human serum albumin (HSA), a multidomain protein, exhibits diverse aggregation behaviors under physiological stress. Here, we investigated how domain-specific ligands (DSLs) such as hemin for domain-I, bilirubin for domain-II, and diazepam for domain-III influences HSA aggregation reactions. Using biophysical techniques including circular dichroism, intrinsic fluorescence, thermal unfolding, quenching assays, red edge excitation shift (REES) analysis, transmission electron microscopy, FTIR, and molecular dynamic simulation (MD-simulation), we show that effect of ligand binding induces distinct aggregation morphologies. Native HSA forms β-sheet-rich worm-like fibrils at ~65 °C and pH 7.4. Hemin binding accelerated aggregation by ~2.5-fold and promoted spherical oligomers, whereas bilirubin slowed it by 2-fold leading to amorphous aggregates, and diazepam produced fibrils similar to native HSA. Ligand binding reduced conformational dynamics and altered equilibrium states, with hemin and bilirubin inducing compactness and internalization of tryptophan residues, thereby decreasing fibril elongation tendency. These findings highlight the role of effect of domain-specific ligands in redesigning aggregation precursor states and pathways. Our study provides mechanistic insights into how small molecules modulate the aggregation landscape of multidomain proteins. By showing that ligands can redirect aggregation toward distinct off-pathway species, this work elucidates the mechanistic basis by which ligand binding modulates protein assembly pathways and aggregates morphology.