Folding subdomains of thioredoxin characterized by native-state hydrogen exchange
Folding subdomains of thioredoxin characterized by native-state hydrogen exchange
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DOI：
10.1110/ps.0239503
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发表时间：
2003-08-01
期刊：
影响因子：
8
通讯作者：
Udgaonkar, JB
中科院分区：
文献类型：
--
作者：
Bhutani, N;Udgaonkar, JB
天然状态的氢交换（HX）的研究，结合NMR光谱，已经进行了大肠杆菌硫氧还蛋白（Trx）的特征的蛋白质的两个折叠亚结构域。骨架酰胺质子交换最慢的只有24个氨基酸残基，总共108个氨基酸残基，可以在pH 7。在24个酰胺氢位点中的每一个处测定导致酰胺氢与溶剂交换的打开事件的自由能（Δ G（op））。属于螺旋α（1）、α（2）和α（4）中残基的酰胺氢的Δ G（op）值与它们仅在完全未折叠状态在天然条件下瞬时取样时与溶剂交换一致。DeltaG（op）值的变性剂依赖性提供了很少的证据表明蛋白质样品部分未折叠形式，其能量低于未折叠状态。属于β链中残基的酰胺氢（形成蛋白质的核心）似乎比属于螺旋中残基的酰胺氢具有更高的DeltaG（op）值，这表明它们可能对交换更稳定。β链对HX的这种明显更高的稳定性可能是因为它们在整体未折叠状态之前的高能中间体中交换出它们的酰胺氢，或者更可能是因为它们在整体未折叠状态中形成残留结构。在这两种情况下，中心β链-β（3），β（2）和β（4）-似乎形成了蛋白质的协同折叠亚基。原生状态HX方法使得有可能表征Trx可以在平衡原生条件下采样的自由能景观。
Native-state hydrogen exchange (HX) studies, used in conjunction with NMR spectroscopy, have been carried out on Escherichia coli thioredoxin (Trx) for characterizing two folding subdomains of the protein. The backbone amide protons of only the slowest-exchanging 24 amino acid residues, of a total of 108 amino acid residues, could be followed at pH 7. The free energy of the opening event that results in an amide hydrogen exchanging with solvent (DeltaG(op)) was determined at each of the 24 amide hydrogen sites. The values of DeltaG(op) for the amide hydrogens belonging to residues in the helices alpha(1), alpha(2), and alpha(4) are consistent with them exchanging with the solvent only when the fully unfolded state is sampled transiently under native conditions. The denaturant-dependences of the values of DeltaG(op) provide very little evidence that the protein samples partially unfolded forms, lower in energy than the unfolded state. The amide hydrogens belonging to the residues in the beta strands, which form the core of the protein, appear to have higher values of DeltaG(op) than amide hydrogens belonging to residues in the helices, suggesting that they might be more stable to exchange. This apparently higher stability to HX of the beta strands might be either because they exchange out their amide hydrogens in a high energy intermediate preceding the globally unfolded state, or, more likely, because they form residual structure in the globally unfolded state. In either case, the central beta strands-beta(3), beta(2), and beta(4)-would appear to form a cooperatively folding subunit of the protein. The native-state HX methodology has made it possible to characterize the free energy landscape that Trx can sample under equilibrium native conditions.