Rawat U, Rao M
Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008, India.
J Biol Chem. 1998 Apr 17;273(16):9415-23. doi: 10.1074/jbc.273.16.9415.
alpha-Crystallin is a multimeric protein that has been shown to function as a molecular chaperone. Present investigations were undertaken to understand its mechanism of chaperoning. For this functional in vitro analysis of alpha-crystallin we used xylose reductase (XR) from Neurospora crassa as the model system. Denaturation studies using the structure-perturbing agent guanidinium chloride indicated that XR folds through a partially folded state that resembles the molten globule. Fluorescence and delay experiments revealed that alpha-crystallin interacts with the molten globule state of XR (XR-m) and prevents its aggregation. Cold lability of alpha-crystallin.XR-m interaction was revealed by temperature shift experiments implicating the involvement of hydrophobic interactions in the formation of the complex. Reconstitution of active XR was observed on cooling the alpha-crystallin.XR-m complex to 4 degrees C or on addition of ATP at 37 degrees C. ATP hydrolysis is not a prerequisite for XR release since the nonhydrolyzable analogue 5'-adenylyl imidodiphosphate (AMP-PNP) was capable of reconstitution of active XR. Experimental evidence has been provided for temperature- and ATP-mediated structural changes in the alpha-crystallin.XR-m complex that shed some light on the mechanism of reconstitution of active XR by this chaperone. The relevance of our finding to the role of alpha-crystallin in vivo is discussed.
α-晶状体蛋白是一种多聚体蛋白，已被证明具有分子伴侣功能。目前开展了相关研究以了解其伴侣机制。为了对α-晶状体蛋白进行这种体外功能分析，我们使用了来自粗糙脉孢菌的木糖还原酶（XR）作为模型系统。使用结构扰动剂氯化胍进行的变性研究表明，XR通过类似于熔球态的部分折叠状态进行折叠。荧光和延迟实验表明，α-晶状体蛋白与XR的熔球态（XR-m）相互作用并防止其聚集。温度变化实验揭示了α-晶状体蛋白与XR-m相互作用的冷不稳定性，这表明疏水相互作用参与了复合物的形成。在将α-晶状体蛋白与XR-m复合物冷却至4℃或在37℃添加ATP时，观察到活性XR的重构。ATP水解不是XR释放的先决条件，因为不可水解的类似物5'-腺苷酰亚胺二磷酸（AMP-PNP）能够重构活性XR。已经提供了实验证据，证明α-晶状体蛋白与XR-m复合物中温度和ATP介导的结构变化，这为该伴侣蛋白重构活性XR的机制提供了一些线索。我们讨论了这一发现与α-晶状体蛋白在体内作用的相关性。
