MOLECULAR MECHANISMS OF ACID DENATURATION - THE ROLE OF HISTIDINE-RESIDUES IN THE PARTIAL UNFOLDING OF APOMYOGLOBIN
MOLECULAR MECHANISMS OF ACID DENATURATION - THE ROLE OF HISTIDINE-RESIDUES IN THE PARTIAL UNFOLDING OF APOMYOGLOBIN
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DOI：
10.1006/jmbi.1994.1257
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发表时间：
1994-04-15
影响因子：
5.6
通讯作者：
BALDWIN, RL
中科院分区：
文献类型：
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作者：
BARRICK, D;HUGHSON, FM;BALDWIN, RL
脱辅基肌红蛋白在接近pH 4时采用部分折叠的中间体构象（I），有时称为熔融球中间体。为了确定哪些组氨酸残基触发这种部分解折叠反应，我们制备了突变体，其中蛋白质中的12个组氨酸残基中的9个被单独取代。然后，我们测量了这些取代蛋白质的酸和尿素诱导的展开曲线。观察到两种酸解折叠转变：天然（N）到中间体（I），以及I到解折叠（U）。这些数据拟合使用一个简单的三态模型，已被证明给酸和尿素诱导的展开野生型脱辅基肌红蛋白的充分描述。其目的是量化N，I和U之间的表观标准吉布斯能差异的变化，以及这些取代导致的展开机制，并测试该模型如何拟合取代蛋白质的数据。在大多数情况下，该模型拟合数据相当好，各种突变体的拟合解折叠参数的显着变化也清楚地显示在原始数据中。得出以下结论。(1)组氨酸24和119在pH 8下协同稳定天然脱辅基肌红蛋白（N），但当pH降低到低于7时一起使N不稳定。(2)组氨酸36在质子化时稳定N。(3)组氨酸取代的血红素结合口袋（残基64，93和97）对N的稳定性几乎没有影响，这表明血红素结合口袋是开放的。(4)组氨酸取代影响N到I的转换，但对I到U的转换几乎没有影响。(5)我们用来描述脱辅基肌红蛋白展开的简单模型不能解释所有的数据，特别是H36Q突变的影响。质子化组氨酸36对稳定N的作用没有包括在模型中，我们认为，当pH从6降低到4时，质子化破坏组氨酸24和119之间的氢键是触发N部分解折叠为I的重要部分，同样，组氨酸24和119之间氢键的形成可能是重折叠过程中从I形成N的动力学过程中的速率决定步骤。
Apomyoglobin adopts a partly folded intermediate conformation (I), sometimes referred to as a molten globule intermediate, near pH 4. To determine which histidine residues trigger this partial unfolding reaction, we made mutants in which nine of the twelve histidine residues in the protein are substituted individually. We then measured acid and urea-induced unfolding curves for these substituted proteins. Two acid unfolding transitions are observed: native (N) to intermediate (I), and I to unfolded (U). These data were fitted using a simple three-state model which has been shown to give an adequate description of acid and urea-induced unfolding of wild-type apomyoglobin. The aim is to quantify changes in the apparent standard Gibbs energy differences between N, I and U, as well as the unfolding mechanism, that result from these substitutions, and to test how well the model fits data for substituted proteins. In most cases, the model fits the data reasonably well, and significant changes in fitted unfolding parameters of various mutants are also clearly visible in the primary data. The following conclusions are drawn. (1) Histidines 24 and 119 synergistically stabilize native apomyoglobin (N) at pH 8, but together destabilize N as pH is decreased below seven. (2) Histidine 36 stabilizes N when it is protonated. (3) Histidine substitutions in the heme-binding pocket (residues 64, 93 and 97) have little effect on the stability of N, suggesting that the heme-binding pocket is open. (4) Histidine substitutions affect the N to I transition but have little effect on the I to U transition. (5) The simple model we use to describe the unfolding of apomyoglobin cannot account for all the data, particularly the effects of the H36Q mutation. The effect of protonated histidine 36 on stabilizing N is not included in the model.We suggest that breaking the hydrogen bond between histidines 24 and 119 by protonation when the pH is decreased from 6 to 4 is an important part of triggering the partial unfolding of N to I, and likewise that formation of the hydrogen bond between histidines 24 and 119 may be a rate-determining step in the kinetic process of forming N from I during refolding.