Exposure to solar UV irradiation damages γ-crystallin, leading to cataract formation via aggregation. α-Crystallin, as a small heat shock protein, efficiently suppresses this irreversible aggregation by selectively binding the denatured γ-crystallin monomer. In this study, liquid chromatography tandem mass spectrometry was used to evaluate UV-325 nm irradiation-induced photodamage of human γD-crystallin in the presence of bovine α-crystallin, atomic force microscope (AFM) and dynamic light scattering (DLS) techniques were used to detect the quaternary structure changes of the α-crystallin oligomer, and Fourier transform infrared spectroscopy and temperature-jump nanosecond time-resolved IR absorbance difference spectroscopy were used to probe the secondary structure changes of bovine α-crystallin. We find that the thermal-induced subunit dissociation of the α-crystallin oligomer involves the breaking of hydrogen bonds at the dimeric interface, leading to three different spectral components at varied temperature regions as resolved from temperature-dependent IR spectra. Under UV-325 nm irradiation, unfolded γD-crystallin binds to the dissociated α-crystallin subunit to form an αγ-complex, then follows the reassociation of the αγ-complex to the partially dissociated α-crystallin oligomer. This prevents the aggregation of denatured γD-crystallin. The formation of the γD-bound α-crystallin oligomer is further confirmed by AFM and DLS analysis, which reveals an obvious size expansion in the reassociated αγ-oligomers. In addition, UV-325 nm irradiation causes a peptide bond cleavage of γD-crystallin at Ala158 in the presence of α-crystallin. Our results suggest a very effective protection mechanism for subunits dissociated from α-crystallin oligomers against UV irradiation-induced aggregation of γD-crystallin, at the expense of a loss of a short C-terminal peptide in γD-crystallin.

暴露于太阳紫外线照射会损害 γ-晶状体蛋白，通过聚集导致白内障形成。α-晶状体蛋白作为一种小型热休克蛋白，通过选择性结合变性的 γ-晶状体蛋白单体，有效抑制这种不可逆的聚集。在本研究中，采用液相色谱串联质谱法评估牛 α-晶状体蛋白存在下 UV-325 nm 照射引起的人 γD-晶状体蛋白的光损伤，原子力显微镜 (AFM) 和动态光散射 (DLS) 技术用于检测α-晶状体蛋白低聚物的四级结构变化，利用傅里叶变换红外光谱和跳温纳秒时间分辨红外吸光差光谱来探测牛α-晶状体蛋白的二级结构变化。我们发现，α-晶状体蛋白低聚物的热诱导亚基解离涉及二聚体界面处氢键的断裂，导致在不同温度区域产生三种不同的光谱成分，如温度依赖性红外光谱所示。在 UV-325 nm 照射下，未折叠的 γD-晶状体蛋白与解离的 α-晶状体蛋白亚基结合形成 αγ-复合物，然后 αγ-复合物与部分解离的 α-晶状体蛋白寡聚物重新结合。这可以防止变性的 γD-晶状体蛋白聚集。AFM 和 DLS 分析进一步证实了 γD 结合的 α-晶状体蛋白寡聚体的形成，这表明重新结合的 αγ-寡聚体的尺寸明显扩大。此外，在 α-晶状体蛋白存在的情况下，UV-325 nm 照射会导致 γD-晶状体蛋白 Ala158 处的肽键断裂。