Nabuurs Sanne M, Westphal Adrie H, van Mierlo Carlo P M
Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands.
J Am Chem Soc. 2009 Feb 25;131(7):2739-46. doi: 10.1021/ja8089476.
During folding of many proteins, molten globules are formed. These partially folded forms of proteins have a substantial amount of secondary structure but lack virtually all tertiary side-chain packing characteristic of native structures. Molten globules are ensembles of interconverting conformers and are prone to aggregation, which can have detrimental effects on organisms. Consequently, molten globules attract considerable attention. The molten globule that is observed during folding of flavodoxin from Azotobacter vinelandii is a kinetically off-pathway species, as it has to unfold before the native state of the protein can be formed. This intermediate contains helices and can be populated at equilibrium using guanidinium hydrochloride as denaturant, allowing the use of NMR spectroscopy to follow molten globule formation at the residue level. Here, we track changes in chemical shifts of backbone amides, as well as disappearance of resonances of unfolded apoflavodoxin, upon decreasing denaturant concentration. Analysis of the data shows that structure formation within virtually all parts of the unfolded protein precedes folding to the molten globule state. This folding transition is noncooperative and involves a series of distinct transitions. Four structured elements in unfolded apoflavodoxin transiently interact and subsequently form the ordered core of the molten globule. Although hydrophobic, tryptophan side chains are not involved in the latter process. This ordered core is gradually extended upon decreasing denaturant concentration, but part of apoflavodoxin's molten globule remains random coil in the denaturant range investigated. The results presented here, together with those reported on the molten globule of alpha-lactalbumin, show that helical molten globules apparently fold in a noncooperative manner.
在许多蛋白质折叠过程中会形成熔球态。这些蛋白质的部分折叠形式具有大量二级结构，但实际上缺乏天然结构所特有的所有三级侧链堆积特征。熔球态是相互转化构象体的集合，易于聚集，这可能对生物体产生有害影响。因此，熔球态引起了相当大的关注。在来自棕色固氮菌的黄素氧还蛋白折叠过程中观察到的熔球态是一种动力学上的非途径物种，因为它必须在蛋白质的天然状态形成之前展开。这种中间体包含螺旋结构，并且可以使用盐酸胍作为变性剂在平衡状态下形成，这使得能够利用核磁共振光谱在残基水平上跟踪熔球态的形成。在这里，我们跟踪随着变性剂浓度降低，主链酰胺化学位移的变化以及未折叠脱辅基黄素氧还蛋白共振信号的消失。数据分析表明，未折叠蛋白质几乎所有部分的结构形成都先于向熔球态的折叠。这种折叠转变是非协同的，涉及一系列不同的转变。未折叠的脱辅基黄素氧还蛋白中的四个结构化元件短暂相互作用，随后形成熔球态的有序核心。尽管色氨酸侧链具有疏水性，但不参与后一过程。随着变性剂浓度降低，这个有序核心逐渐扩展，但在所研究的变性剂范围内，黄素氧还蛋白熔球态的一部分仍保持无规卷曲。此处呈现的结果，连同关于α-乳白蛋白熔球态的报道结果，表明螺旋熔球态显然以非协同方式折叠。
