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首页> 外文期刊>Journal of Molecular Biology >Kinetic and equilibrium intermediate states are different in LYLA1, a chimera of lysozyme and alpha-lactalbumin.
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Kinetic and equilibrium intermediate states are different in LYLA1, a chimera of lysozyme and alpha-lactalbumin.

机译：LYLA1是溶菌酶和α-乳清蛋白的嵌合体，其动力学和平衡中间状态不同。

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For several proteins, a striking resemblance has been observed between the equilibrium partially folded state and the kinetic burst-phase intermediate, observed just after the dead-time in refolding experiments. This has led to the general statement that the conformation of both types of intermediates is similar. We show, at least for one of the proteins investigated here, that, although both states have some common characteristics, they are not identical. LYLA1 is a chimeric protein resulting from the transplantation of the Ca(2+)-binding loop and the adjacent helix C of bovine alpha-lactalbumin into the homologous position (residues 76-102) in human lysozyme. The apo-form of LYLA1 unfolds through a partially folded state, in analogy with the folding behaviour of the structurally homologous alpha-lactalbumin. The folding kinetics of LYLA1 and of its wild-type homologue, human lysozyme, are investigated by means of stopped-flow fluorescence and CD spectroscopy. In the case of human lysozyme, refolding involves parallel pathways as indicated by experiments in the presence of a fluorescent inhibitor. For apo-LYLA1, the burst-phase intermediate is compared with the equilibrium intermediate. At neutral pH, both states correspond, in that an important amount of secondary structure has been established, but the burst-phase intermediate is shown to be significantly less stable than the equilibrium intermediate. At pH 1.85, in the presence of 1.5 M guanidinium hydrochloride (GdnHCl) and at 25 degrees C, the equilibrium partially folded state of LYLA1 is 100% populated. When LYLA1 is rapidly diluted from 6 M GdnHCl to 1.5 M under these conditions, a time-dependent evolution of the fluorescence signal is observed, reflecting the transition from a burst-phase to a different equilibrium intermediate. These results provide strong evidence for the non-identity of both states in this protein. Copyright 1999 Academic Press.
机译：对于几种蛋白质，在重新折叠实验中的死时间之后，观察到在平衡的部分折叠状态和动力学猝发相中间体之间的惊人相似。这导致人们普遍认为两种中间体的构象相似。我们显示，至少对于这里研究的一种蛋白质，尽管这两种状态都有一些共同的特征，但它们并不相同。 LYLA1是一种嵌合蛋白，由Ca（2+）结合环和牛α-乳清蛋白的相邻螺旋C移植到人溶菌酶的同源位置（残基76-102）中产生。与结构同源的α-乳清蛋白的折叠行为类似，LYLA1的脱辅基形式通过部分折叠状态展开。 LYLA1及其野生型同源物（人类溶菌酶）的折叠动力学通过停止流荧光和CD光谱研究。在人溶菌酶的情况下，重折叠涉及在荧光抑制剂存在下的实验所表明的平行途径。对于载脂蛋白LYLA1，将猝发相中间体与平衡中间体进行比较。在中性pH下，两种状态都相对应，因为已经建立了大量的二级结构，但是爆裂相中间体的稳定性明显低于平衡中间体。在pH 1.85下，在存在1.5 M盐酸胍（GdnHCl）和25°C的条件下，LYLA1的平衡部分折叠状态达到100％。当在这些条件下将LYLA1从6 M GdnHCl快速稀释至1.5 M时，观察到荧光信号随时间的变化，反映了从猝发相到不同平衡中间体的跃迁。这些结果为该蛋白质中两种状态的不一致性提供了有力的证据。版权所有1999，学术出版社。

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