Marmorino J L, Lehti M, Pielak G J
Department of Chemistry, University of North Carolina at Chapel Hill 27599-3290, USA.
J Mol Biol. 1998 Jan 16;275(2):379-88. doi: 10.1006/jmbi.1997.1450.
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, delta GA reversible D and delta GN reversible 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 delta GA reversible D versus-delta GN reversible D plot 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 approximately 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.
A态是一种平衡物种，被认为代表熔球态，即一种具有天然二级结构和非天然、波动三级结构的蛋白折叠中间态。我们使用酵母异-1-铁细胞色素c来测试其A态中一种进化不变的三级相互作用。通过圆二色性（CD）分光偏振法监测的热变性用于确定A态和天然态的稳定性，即ΔGA可逆D和ΔGN可逆D。我们研究了野生型蛋白、在N端和C端螺旋之间界面处有取代的七个变体以及四个对照变体。对照变体与界面变体具有相同的氨基酸变化，但这些变化靠近而非位于界面处。我们还研究了pH值和硫酸盐浓度的依赖性，发现虽然这些因素会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和变体之间的稳定性差异。界面变体的ΔGA可逆D对ΔGN可逆D图的斜率接近1，对照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态和天然态的稳定程度相同，证实了我们的初步报告。我们确定A态变性的热容变化约为天然态变性值的60%，表明A态内部类似天然态。我们结合铁细胞色素c的折叠动力学讨论了我们的结果。
