From the Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands.
From the Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands.
J Biol Chem. 2010 Feb 5;285(6):4165-4172. doi: 10.1074/jbc.M109.087932. Epub 2009 Dec 3.
Kinetic intermediates that appear early during protein folding often resemble the relatively stable molten globule intermediates formed by several proteins under mildly denaturing conditions. Molten globules have a substantial amount of secondary structure but lack virtually all tertiary side-chain packing characteristics of natively folded proteins. Due to exposed hydrophobic groups, molten globules are prone to aggregation, which can have detrimental effects on organisms. The molten globule that is observed during folding of alpha-beta parallel flavodoxin from Azotobacter vinelandii is a remarkably non-native species. This folding intermediate is helical and contains no beta-sheet and is kinetically off-pathway to the native state. It can be trapped under native-like conditions by substituting residue Phe(44) for Tyr(44). To characterize this species at the residue level, in this study, use is made of interrupted hydrogen/deuterium exchange detected by NMR spectroscopy. In the molten globule of flavodoxin, the helical region comprising residues Leu(110)-Val(125) is shown to be better protected against exchange than the other ordered parts of the folding intermediate. This helical region is better buried than the other helices, causing its context-dependent stabilization against unfolding. Residues Leu(110)-Val(125) thus form the stable core of the helical molten globule of alpha-beta parallel flavodoxin, which is almost entirely structured. Non-native docking of helices in the molten globule of flavodoxin prevents formation of the parallel beta-sheet of native flavodoxin. Hence, to produce native alpha-beta parallel protein molecules, the off-pathway species needs to unfold.
在蛋白质折叠过程中早期出现的动力学中间体通常类似于在轻度变性条件下形成的几个蛋白质的相对稳定的无规卷曲中间体。无规卷曲具有大量的二级结构，但几乎缺乏天然折叠蛋白质的所有三级侧链堆积特征。由于暴露的疏水区，无规卷曲容易聚集，这可能对生物体产生不利影响。在来自 Azotobacter vinelandii 的α-β平行黄素蛋白折叠过程中观察到的无规卷曲是一种非常非天然的物种。这种折叠中间体是螺旋的，不包含β-折叠，并且在动力学上偏离了天然状态。通过将残基 Phe(44)取代为 Tyr(44)，可以在类似天然的条件下捕获这种折叠中间体。为了在残基水平上表征这种物种，在本研究中，使用 NMR 光谱检测到的中断氢/氘交换来进行。在黄素蛋白的无规卷曲中，包含残基 Leu(110)-Val(125)的螺旋区域相对于折叠中间体的其他有序部分，显示出对交换的更好保护。该螺旋区域比其他螺旋更好地埋藏，导致其对展开的上下文相关稳定。因此，残基 Leu(110)-Val(125)形成α-β平行黄素蛋白的稳定螺旋无规卷曲核心，几乎完全有结构。黄素蛋白无规卷曲中螺旋的非天然对接阻止了天然黄素蛋白的平行β-折叠的形成。因此，为了产生天然的α-β平行蛋白质分子，需要使偏离途径的物种展开。
