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

β-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 β-crystallins naturally assemble into various heteroge-neous oligomers with different sizes. Here, we systematically investigated the thermal sta-bility of the different secondary structures present in β-crystallins and then the dynamic process for the thermal-induced unfolding of β-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 β-sheets in β-crystallin are the most unstable with a transition midpoint temperature at 36.0±2.1 oC, 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 β-crystallin oligomers with a transition midpoint tempera-ture of 40.4±0.7 oC. The global unfolding of β-crystallins that leads to denaturation and aggregation indicated by the formation of intermolecular anti-parallel β-sheets has a transi-tion midpoint temperature determined as 72.4±0.2 oC. Temperature-jump time-resolved IR absorbance difference spectroscopy analysis further reveals that thermal-induced unfolding of β-crystallins occurs firstly in the anti-parallel β-sheets in the N-terminal domains with a time constant of 50 ns