【摘要】 通过变温傅里叶变换红外光谱和脉冲升温时间分辨红外光谱,系统地研究了β晶状体蛋白不同二级结构的热稳定性,以及热诱导β晶状体蛋白去折叠的动力学过程.结果表明,β晶状体蛋白N端的β反平行折叠热稳定性最低(转变的中点温度为36.0±2.1℃),该结构的破坏导致了一种富含无规卷曲结构的β晶状体蛋白中间体形成,认为该中间体可能由β晶状体寡聚体解聚后形成的单体,单体形成的中点温度为40.4±7℃.β晶状体蛋白全局去折叠引发蛋白变性聚集的转变的中点温度为72.4±0.2℃.脉冲升温时间分辨红外光谱进一步揭示出β晶状体蛋白的热诱导去折叠开始于N端的β反平行折叠结构,该结构去折叠所需时间约为50 ns.更多还原

【Abstract】 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 heterogeneous oligomers with different sizes.Here,we systematically investigated the thermal stability 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 ℃,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 temperature of 40.4±0.7 ℃.The global unfolding of β-crystallins that leads to denaturation and aggregation indicated by the formation of intermolecular anti-parallel β-sheets has a transition midpoint temperature determined as 72.4±0.2 ℃.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.更多还原