Detection and characterization of alpha-crystallin intermediate with maximal chaperone-like activity
Detection and characterization of alpha-crystallin intermediate with maximal chaperone-like activity
复制标题
DOI：
10.1006/bbrc.1997.6950
复制
发表时间：
1997-07-18
影响因子：
3.1
通讯作者：
Liang, JJN
中科院分区：
文献类型：
--
作者：
Das, BK;Liang, JJN
据报道，晶状体α-晶体蛋白的作用类似于伴侣分子，通过部分展开增强了伴侣般的活性。然而，部分去折叠的本质还没有完全被理解，在这个项目中，我们研究了α-晶体蛋白在盐酸胍(GdnHCl)中的去折叠和复性过程。色氨酸荧光(三级结构)和远紫外圆二色谱(UVCD)(二级结构)表明在去折叠过程中存在中间体，ANS(1-苯胺基-8-萘磺酸)荧光清楚地表明去折叠过程中存在两步转变，并表明α-晶状体蛋白的疏水性在0.8-1.0M GdnHCl时达到最大值。这种α-晶体蛋白中间体似乎处于熔融的球状状态；近、远UVCD测量的构象研究表明，α-晶体蛋白中间体具有与天然蛋白质显著不同的三级结构，但具有几乎相同的二级结构。FPLC尺寸排除层析显示，中间体的四级结构(聚集体的大小)也与天然蛋白保持不变。在去折叠复性途径中，α-晶状体蛋白中间体的最大疏水性伴随着β(H)-晶状体蛋白对聚集的最大保护。然而，部分展开的一个不利影响是，α-晶状体蛋白在高浓度下形成中间聚集体。总之，这些结果清楚地证明了α-晶体蛋白中间体的生物学意义：它是一种比天然α-晶体蛋白更有效的伴侣。(C)1997年学术出版社。
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 beta(H)-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. (C) 1997 Academic Press.