Comparison between the unfolding rate and structural fluctuations in native lysozyme—effects of denaturants, ligand binding, and intrachain cross‐linking on hydrogen exchange and unfolding kinetics
Comparison between the unfolding rate and structural fluctuations in native lysozyme—effects of denaturants, ligand binding, and intrachain cross‐linking on hydrogen exchange and unfolding kinetics
复制标题
天然溶菌酶的解折叠速率和结构波动的比较——变性剂、配体结合和链内交联对氢交换和解折叠动力学的影响
作者：
S. Segawa;K. Kume
通过红外光谱中酰胺II带强度的变化，研究了溶菌酶中肽NH基团之间的氢交换反应。参与分子内氢键的最慢的交换氢原子在较低的温度下进一步分为两组，其中一半通过局部去折叠交换，另一半通过主要合作去折叠交换。为了研究氢交换率的变化与去折叠速率常数变化的相关性，我们通过局部去折叠观察了链内交联、变性剂的添加和配体结合对交换率的影响。虽然Glu 35和Trp 108的链内交联会大大降低主要去折叠的交换率(1/22000)，但局部去折叠的交换率仅略有下降(1/20)。即使在更高的温度下，大多数完整的溶菌酶分子展开，交联型溶菌酶的折叠构象仍然保持致密，并且不存在许多侧链原子松散堆积的中间体，从而使氢交换反应迅速发生。2-Prod分子的添加和(NAG)3的结合都不会通过局部展开影响交换率。我们的实验证实，解折叠速率常数的变化与肽氢交换所通过的相对灵活的氢键结构的波动变化无关。
The hydrogen‐exchange reactions of peptide NH groups in lysozyme were studied by the change in the intensity of the amide II band in the ir spectrum. The slowest exchanging hydrogens, which are involved in intramolecular hydrogen bonding, are further divided into two groups at lower temperatures; half of them are exchanged through local unfolding and the other half through major cooperative unfolding. In order to study the correlation of the change in hydrogen‐exchange rates with the change in the unfolding rate constant, we observed the effects of intrachain cross‐linking, the addition of denaturant and ligand binding on the exchange rates through local unfolding. Although the exchange rate through major unfolding is greatly decreased by intrachain cross‐linking between Glu 35 and Trp 108 (1/22000), the exchange rate through local unfolding is only slightly decreased (1/20). Even at higher temperatures, where most intact lysozyme molecules unfold, the folded conformation of cross‐linked lysozyme remains compact, and no intermediate exists in which many side‐chain atoms are packed loosely so that the hydrogen‐exchange reaction occurs rapidly. Neither the addition of 2‐PrOD molecules nor (NAG)3 binding affects the exchange rates through local unfolding. Our experiments confirm that the change in the unfolding rate constant does not correlate with the change in fluctuations in the relatively flexible hydrogen‐bonded structure through which the exchange of peptide hydrogens takes place.
胰蛋白酶抑制剂中的氢交换率与尿素的热稳定性不相关。
影响因子：
2.9
作者：
Hilton,BD;Trudeau,K;Woodward,CK
通讯作者：
Woodward,CK
溶菌酶中单个色氨酸吲哚 NH 氢的核磁共振研究蛋白质中氢交换的机制。
影响因子：
2.9
作者：
Wedin,RE;Delepierre,M;Dobson,CM;Poulsen,FM
通讯作者：
Poulsen,FM