Heat-induced conformational change and increased chaperone activity of lens alpha-crystallin
Heat-induced conformational change and increased chaperone activity of lens alpha-crystallin
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DOI：
10.1076/ceyr.16.4.303.10691
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发表时间：
1997-04-01
影响因子：
2
通讯作者：
Chakrabarti, B
中科院分区：
文献类型：
--
作者：
Das, BK;Liang, JJN;Chakrabarti, B
目的。 α-晶状体蛋白是眼晶状体的主要结构蛋白，已知具有类似伴侣的活性。我们的目标是阐明 α-晶状体蛋白在 60 摄氏度下经历的热转变的性质以及这种转变对伴侣活性的影响。方法。使用 FPLC 尺寸排阻色谱、远紫外和近紫外圆二色性以及色氨酸 (Trp) 和 1-苯胺基-8-萘磺酸盐 (ANS) 荧光来研究构象变化。使用二硫苏糖醇 (DTT) 降低的胰岛素的浊度来研究伴侣活性。结果。热转变被确定为主要是三级（部分展开）和四级高分子量（HMW）聚集结构的构象变化，以及二级结构（β-折叠）损失10个百分点。三级结构的初始部分扰动增加了伴侣活性，但 HMW 聚集体的增加较小。在体内形成的 HMW α-晶状体蛋白中观察到类似的结果。结论。在 60 摄氏度下观察到的 α-晶状体蛋白的构象变化和 HMW 聚集，以及体内形成的 HMW 聚集体，增加了分子伴侣的活性。
Purpose. Alpha-crystallin is the major structural protein of the eye lens known to have chaperone-like activity. Our objective is to elucidate the nature of the thermal transition that alpha-crystallin undergoes at 60 degrees C and the effect of this transition on the chaperone activity.Methods. FPLC size exclusion chromatography, far- and near-ultraviolet circular dichroism, and tryptophan (Trp) and 1-anilino-8-naphthalenesulfonate (ANS) fluorescence were used to study conformational change. Turbidity of dithiothreitol (DTT)-reduced insulin was used to study chaperone activity.Results. The thermal transition was identified as a conformational change in mainly tertiary (partial unfolding) and quaternary high-molecular-weight (HMW) aggregation structures, along with a loss of 10 percentage points of secondary structure (beta-sheet). Initial partial perturbation in tertiary structure increased chaperone activity, but the increase was less in the HMW aggregate. Similar results were observed in in vivo-formed HMW alpha-crystallin.Conclusions. The conformational change and HMW aggregation of alpha-crystallin observed at 60 degrees C, as well as in vivo-formed HMW aggregates, increased chaperone activity.