Native tertiary structure in an A-state.
Native tertiary structure in an A-state.
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A 状态的天然三级结构。
DOI：
10.1006/jmbi.1997.1450
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发表时间：
1998
期刊：
影响因子：
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通讯作者：
Pielak,GJ
中科院分区：
文献类型：
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作者：
Marmorino,JL;Lehti,M;Pielak,GJ
a态是一种平衡态，被认为代表熔融球，一种具有天然二级结构和非天然波动三级结构的通路蛋白折叠中间体。我们使用酵母iso-1-铁胞色素c来测试其a态的进化不变的三级相互作用。采用圆二色性（CD）光谱偏振法监测热变性，以确定当下a态和原生态稳定性ΔGA和ΔGN当下D。我们检测了野生型蛋白，在N端和c端螺旋之间的界面上有7个替换的变体，以及4个控制变体。控制组的氨基酸变化与界面变异体相同，但变化是靠近界面而不是在界面上。我们还研究了pH和硫酸盐浓度依赖性，发现虽然这些因素影响最不稳定变体的远紫外CD光谱，但它们不会改变野生型蛋白质和变体之间的稳定性差异。ΔGA当下一种界面变体的坡度接近统一，而控制变体具有接近野生型的稳定性。这些结果表明，螺旋-螺旋相互作用对a态和原生态的稳定程度相同，证实了我们的初步报道。我们确定了a态变性的热容变化是原生态变性值的约60%，表明a态内部与原生态相似。我们讨论了我们的结果与铁细胞色素c折叠动力学的关系。
The A-state is an equilibrium species that is thought to represent the molten globule, an on-pathway protein folding intermediate with native secondary structure and non-native, fluctuating tertiary structure. We used yeast iso-1-ferricytochrome c to test for an evolutionary-invariant tertiary interaction in its A-state. Thermal denaturation monitored by circular dichroism (CD)spectropolarimetry was used to determine A-state and native-state stabilities, ΔGA⇄Dand ΔGN⇄D. We examined the wild-type protein, seven variants with substitutions at the interface between the N and C-terminal helices, and four control variants. The controls have the same amino acid changes as the interface variants, but the changes are close to, not at, the interface. We also examined the pH and sulfate concentration dependencies and found that while these factors affect the far-UV CD spectra of the least stable variants, they do not alter the difference in stability between the wild-type protein and the variants. A ΔGA⇄Dversus- ΔGN⇄Dplot for the interface variants has a slope near unity and the control variants have near-wild-type stability. These results show that the helix-helix interaction stabilizes the A-state and the native state to the same degree, confirming our preliminary report. We determined that the heat capacity change for A-state denaturation is ≈60% of the value for native-state denaturation, indicating that the A-state interior is native-like. We discuss our results in relation to ferricytochrome c folding kinetics.