Thermal-induced Unfolding of β-Crystallin and Disassembly of its Oligomers Revealed by Temperature-Jump Time-Resolved Infrared Spectroscopy

beta-Crystallins are the major structural proteins existing in the vertebrate lens, and their conformational stability is critical in maintaining the life-long transparency and refraction index of the lens. Seven subunits of beta-crystallins naturally assemble into various heterogeneous oligomers with different sizes. Here, we systematically investigated the thermal stability of the different secondary structures present in beta-crystallins and then the dynamic process for the thermal-induced unfolding of beta-crystallins by Fourier transform infrared spectroscopy-monitored thermal titration and temperature-jump nanosecond time-resolved IR difference absorbance spectra. Our results show that the N-terminal anti-parallel beta-sheets in beta-crystallin are the most unstable with a transition midpoint temperature at 36.0 +/- 2.1 degrees C, leading to the formation of an intermediate consisting vastly of random coil structures. This intermediate structure is temporally assigned to that of the monomer generated by the thermal-induced disassembly of beta-crystallin oligomers with a transition midpoint temperature of 40.4 +/- 0.7 degrees C. The global unfolding of beta-crystallins that leads to denaturation and aggregation indicated by the formation of intermolecular anti-parallel beta-sheets has a transition midpoint temperature determined as 72.4 +/- 0.2 degrees C. Temperature-jump time-resolved IR absorbance difference spectroscopy analysis further reveals that thermal-induced unfolding of beta-crystallins occurs firstly in the anti-parallel beta-sheets in the N-terminal domains with a time constant of 50 ns.