Spatial relationship between the prodan site, Trp-214, and Cys-34 residues in human serum albumin and loss of structure through incremental unfolding

Abstract

Prodan (6-propionyl-2-(dimethylamino)-naphthalene), a competitive inhibitor of warfarin binding to human serum albumin (HSA) at drug site I, was used to determine the inter- and intradomain distances of HSA. The fluorescence resonance energy transfer (FRET) distances between prodan and Trp-214, prodan and 7-(diethyl amino)-4-methylcoumarin 3-maleimide (CM)-modified Cys-34, and Trp-214 and CM-Cys-34 were determined to be 25.5±0.5Å, 33.1±0.8Å, and 32.4±1Å, respectively. FRET analysis showed that low concentration of palmitic acid (5μM) increased the interdomain distance between the Trp-214 in domain II and CM-Cys-34 in domain I by 5Å without perturbing the secondary structure of HSA and the immediate environment of Trp-214. Palmitic acid (5μM) increased the prodan fluorescence by increasing the quantum yield of bound prodan without altering the tryptophan environment. However, palmitic acid (>10μM) decreased the prodan fluorescence and increased the tryptophan fluorescence. Our results indicate that the high affinity palmitic acid binding site is located at the interface of domains I and II. On the basis of our measurements, a schematic model representing the drug site-1, Trp-214, and Cys-34 along with the palmitic acid sites has been constructed. In addition, prodan fluorescence, FRET, and ligand binding were used to monitor guanidine hydrochloride-induced denaturation of HSA. An analysis of the equilibrium unfolding data suggests that HSA undergoes a two-state unfolding transition with no detectable intermediate. However, kinetic analysis using multiple probes and thermal denaturation studies showed that the unfolding of the prodan site in HSA preceded the unfolding of tryptophan environment. In addition, the separation of domain I and II occurred before the global unfolding of the protein. The data support the idea that HSA loses its structure incrementally during its unfolding.