Effect of H helix destabilizing mutations on the kinetic and equilibrium folding of apomyoglobin
Effect of H helix destabilizing mutations on the kinetic and equilibrium folding of apomyoglobin
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DOI：
10.1006/jmbi.1998.2273
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发表时间：
1999-01-08
影响因子：
5.6
通讯作者：
Wright, PE
中科院分区：
文献类型：
--
作者：
Cavagnero, S;Dyson, HJ;Wright, PE
酸变性的脱辅基肌红蛋白（apoMb）在多肽区域中含有残留的螺旋结构，其对应于折叠蛋白质的H螺旋。为了阐明这种残余的二级结构在蛋白质折叠过程中的作用，并确定是否在变性状态下的残余结构影响整体折叠速率或形成的突发阶段中间体的速率，我们已经研究了平衡和动力学折叠行为的突变体的设计，以破坏残余的二级结构中的H螺旋区域。Asn132和Glu136都被改变为Gly（N132G，E136G）以实现这种去稳定化。圆二色光谱表明，突变蛋白含有较少的螺旋结构在酸变性状态，并在平衡中间状态，在pH 4.2比野生型蛋白。这两种蛋白质的天然状态的CD光谱几乎相同。每个物种的重折叠动力学进行了测量，在远紫外区的停流CD和NMR猝灭流脉冲标记。在相同的条件下，CD检测到的野生型和突变型脱辅基肌红蛋白从酸变性状态或从尿素变性状态的重折叠发生在非常相似的速率后，爆发阶段发生得太快，无法通过停流技术测量。尿素依赖性的展开和重折叠速率是一致的存在下，至少有一个强制性的途径中间体在野生型和突变体蛋白。突变蛋白的动力学中间体比野生型蛋白的动力学中间体稳定性低得多。氢交换脉冲标记实验表明，与野生型蛋白相比，H螺旋在突变体的突发期重折叠过程中不稳定，但在较慢的阶段中变得稳定。虽然野生型和突变体蛋白都形成紧凑的中间体，但它们在二级结构的内容和位置上不同。AGH亚结构域的折叠速率，发生在过渡态之前，对于N132G，E136G突变蛋白质来说是相当慢的。在H螺旋区域自发形成螺旋结构的强烈倾向不是有效折叠的先决条件，也不是形成平衡或动力学中间体的先决条件。这些观察结果表明，虽然通过强制性中间体进行折叠的脱辅基肌红蛋白，该中间体的精确结构并不重要，其二级结构可能会改变，而不会显著影响整体重折叠动力学或最终折叠状态的完整性。(C)北京：科学出版社.
Acid-denatured apomyoglobin (apoMb) contains residual helical structure in the region of the polypeptide which corresponds to the H helix of the folded protein. In order to elucidate the role of this residual secondary structure in the protein folding process and to determine whether residual structure in the denatured state affects either the overall rate of folding or the rate of formation of a burst phase intermediate, we have examined the equilibrium and kinetic folding behavior of a mutant designed to destabilize residual secondary structure in the H helix region. Both Asn132 and Glu136 were changed to Gly (N132G,E136G) to effect this destabilization. Circular dichroism spectra show that the mutant protein contains less helical structure in the acid-denatured state and in the equilibrium intermediate state at pH 4.2 than does the wild-type protein. The CD spectra of the native states of the two proteins are nearly identical. The refolding kinetics for each of the species were measured by stopped-flow CD in the far-UV region and by NMR quench-flow pulse labeling. Under identical conditions, the CD-detected refolding of wild-type and mutant apomyoglobin from the acid-denatured state or from the urea-denatured state occurs at very similar rates following a burst phase that occurs too rapidly to measure by the stopped-flow technique. The urea dependence of the unfolding and refolding rates is consistent with the presence of at least one obligatory on-pathway intermediate in both wild-type and mutant proteins. The kinetic intermediate of the mutant protein is considerably less stable than that of the wild-type protein. Hydrogen exchange pulse labeling experiments indicate that, in contrast to the wild-type protein, the H helix is not stabilized during the burst phase refolding of the mutant but becomes stabilized during the slower phases. While the wild-type and mutant proteins both form compact intermediates, these differ in the content and location of secondary structure. The rate of folding of the AGH subdomain, which takes place prior to the transition state, is substantially slower for the N132G,E136G mutant protein. A strong propensity for spontaneous formation of helical structure in the H helix region is not a prerequisite for efficient folding nor for formation of equilibrium or kinetic intermediates. These observations suggest that while folding of apomyoglobin proceeds through an obligatory intermediate, the precise structure of this intermediate is not critical and its secondary structure may be altered without substantially affecting either the overall refolding kinetics or the integrity of the final folded state. (C) 1999 Academic Press.