Abstract
α-Synuclein (α-Syn) is implicated in the pathophysiology of Parkinson's disease (PD) and plays a significant role in neuronal degeneration. Iron response proteins (IRPs) bind to iron response elements (IREs) found in the 5'-untranslated regions (5'-UTRs) of the messenger RNA that encode the α-Syn gene. This study used multi-spectroscopic approach techniques to investigate the impact of iron on α-Syn IRE RNA binding to IRP1. The formation of a stable complex between α-Syn RNA and IRP1 was suggested by fluorescence quenching results. Fluorescence measurements showed that α-Syn RNA and IRP1 had a strong interaction, with a binding constant (K(a)) of 21.0 × 10(6) M(-1) and 1:1 binding stoichiometry. About one binding site per IRP1 molecule was suggested by the α-Syn RNA binding. The K(a) for α-Syn RNA•IRP1 with added Fe(2+) (50 μM) was 6.4 μM(-1). When Fe(2+) was added, the K(a) of α-Syn RNA•IRP1 was reduced by 3.3 times. These acquired K(a) values were used to further understand the thermodynamic characteristics of α-Syn RNA•IRP1 interactions. The thermodynamic properties clearly suggested that α-Syn RNA binding to IRP1 was an entropy-favored and enthalpy-driven event, with significant negative ΔH and small positive ΔS. For α-Syn RNA•IRP1, the Gibbs free energy (ΔG) was -43.7 ± 2.7 kJ/mol, but in the presence of Fe(2+), it was -36.3 ± 2.1 kJ/mol. These thermodynamic calculations indicated that hydrogen bonding as well as van der Waals interactions might help to stabilize the complex formation. Additionally, far-UV CD spectra verified α-Syn RNA•IRP1 complex formation, and α-Syn RNA and Fe(2+) induce secondary structural alteration of IRP1. According to our findings, iron alters the hydrogen bonding in α-Syn RNA•IRP1 complexes and induces a structural change in IRP1. This suggests that iron selectively affects the thermodynamics of these RNA-protein interactions.