Revisiting Structural Hieracrchy: A Fluorescence Investigation of Unfolding of an Oligomeric Protein

Abstract

α-crystallin is a multimeric lens protein with chaperone-like function and is responsible for maintaining lens transparency. The structural stability and unfolding-refolding properties of this protein are believed to be important for its function. We undertook a multi-probe based fluorescence approach to explore the changes in the various levels of organization of α-crystallin at different urea concentration region. Steady-state fluorescence emission and quenching studies using extrinsic and intrinsic fluorescence probes reveal that at 0.6 M urea a compact structural intermediate is formed which has a native-like secondary structure with minimal yet detectable tertiary structure perturbation associated with enhanced surface exposure of hydrophobic groups, possibly arising from interfacial structure meltdown. At 2.8 M urea the tertiary interactions undergo almost complete collapse with partial disintegration of secondary and quaternary structure. Investigation of surface solvation with picosecond resolved fluorescence transients of acrylodan covalently tagged to α-crystallin reveals a dry native-like core of α-crystallin at 0.6 M urea compared to enhanced water penetration at 2.8 M urea and extensive solvation at 6 M urea. Temperature dependent subunit exchange kinetics reveal decrease of activation energy for the subunit exchange process by 22 kJ mol-1 on changing urea concentration from 0 to 0.6 M compared to over 75 kJ mol-1 on changing urea concentration from 0 to 2.8 M. Dynamic light scattering study indicates swelling at 0.6 M urea, however oligomerization is retained as observed from sedimentation equilibrium experiment. At 2.8 M urea the oligomeric size is reduced and a monomer is produced at 6 M urea. These data clearly demonstrates that tertiary structure dissolution precedes oligomeric degradation. Such non-hierarchical structure dissolution indicates the possibility of tertiary contact formation to be a rather later folding event in case of large oligomeric proteins likes α-Crystallin.