Das B K, Liang J J
Center for Ophthalmic Research, Brigham and Women's Hospital, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Biochem Biophys Res Commun. 1997 Jul 18;236(2):370-4. doi: 10.1006/bbrc.1997.6950.
Lens alpha-crystallin has been reported to act like a chaperone molecule, with the chaperone-like activity enhanced by partial unfolding. The nature of the partial unfolding, however, is not fully understood. In this project, the unfolding and refolding process of alpha-crystallin was studied with guanidine hydrochloride (GdnHCl). Trp fluorescence (tertiary structure) and far-ultraviolet circular dichroism (UVCD) (secondary structure) demonstrated the presence of an intermediate in the unfolding pathway. ANS (1-anilino-8-naphthalenesulfonate) fluorescence clearly indicated a two-step transition in the unfolding-refolding process and showed that maximum hydrophobicity of the alpha-crystallin occurred at 0.8-1.0 M GdnHCl. This alpha-crystallin intermediate appears to be in a molten globule state; conformational study by near- and far-UVCD measurements indicated that alpha-crystallin intermediate exhibited tertiary structure which was significantly altered from that of the native protein, but had nearly the same secondary structure. Quaternary structure (size of aggregate) of the intermediate also remained unchanged from that of the native protein, as shown by FPLC size exclusion chromatography. The maximal hydrophobicity of the alpha-crystallin intermediate in the unfolding-refolding pathway was accompanied by maximal protection of betaH-crystallin from aggregation. However, an adverse effect of partial unfolding is that the alpha-crystallin intermediate aggregates at high concentrations. Together, these results clearly demonstrated the biological significance of the alpha-crystallin intermediate: it is a more effective chaperone than native alpha-crystallin.
据报道，晶状体α-晶体蛋白具有伴侣分子的作用，其伴侣样活性通过部分解折叠而增强。然而，部分解折叠的本质尚未完全了解。在本项目中，利用盐酸胍（GdnHCl）研究了α-晶体蛋白的解折叠和重折叠过程。色氨酸荧光（三级结构）和远紫外圆二色性（UVCD）（二级结构）表明在解折叠途径中存在一个中间体。ANS（1-苯胺基-8-萘磺酸盐）荧光清楚地表明了解折叠-重折叠过程中的两步转变，并表明α-晶体蛋白的最大疏水性出现在0.8 - 1.0 M GdnHCl时。这种α-晶体蛋白中间体似乎处于熔球状态；通过近紫外和远紫外CD测量进行的构象研究表明，α-晶体蛋白中间体表现出三级结构，与天然蛋白质的三级结构有显著改变，但二级结构几乎相同。如FPLC尺寸排阻色谱所示，中间体的四级结构（聚集体大小）也与天然蛋白质保持不变。在解折叠-重折叠途径中，α-晶体蛋白中间体的最大疏水性伴随着对βH-晶体蛋白聚集的最大保护作用。然而，部分解折叠的一个不利影响是α-晶体蛋白中间体在高浓度下会聚集。总之，这些结果清楚地证明了α-晶体蛋白中间体具有生物学意义：它是一种比天然α-晶体蛋白更有效的伴侣分子。
