Surface plasmon resonance study of the interaction of 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt (bis-ANS) and adenosine triphosphate (ATP) with oligomeric recombinant human lens αA-crystallin

Abstract

α-Crystallin, an abundant mammalian lens protein made up of two subunits (αA- and αB-crystallin), is involved in the maintenance of the optimal refractive index in the lens. The protein is implicated in the pathophysiology of a large number of retinal diseases including cataract, age-related macular degeneration, diabetic retinopathy, and uveitis. α-Crystallin belongs to the small heat shock protein (sHSP) family, forms large oligomeric structures, and functions as a molecular chaperone appearing very early during embryonic development. To gain mechanistic insight into the structural and functional role of α-crystallin and its alterations in various retinal diseases, it is important to study the interaction chemistry with its known partners. The hydrophobic sites in α-crystallin have been studied extensively using environmentally sensitive fluorescent probes such as 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt (bis-ANS) that interacts with both subunits of α-cystallin in 1:1 stoichiometry at 37 °C and diminishes the chaperone-like activity of the protein. Furthermore, it has been shown that ATP plays a crucial role in the association of α-crystallin with substrate proteins. We use surface plasmon resonance (SPR) to monitor the interactions of immobilized oligomeric recombinant αA subunit of human α-crystallin protein with bis-ANS and ATP. We assess the thermodynamic parameters and kinetics of such interactions at various temperatures. Our results indicate that bis-ANS binds to αA-crystallin with higher affinity when compared with ATP, although both αA-crystallin and αB-crystallin display fast interaction kinetics.