KINETICS OF HYDROGEN-BOND BREAKAGE IN THE PROCESS OF UNFOLDING OF RIBONUCLEASE-A MEASURED BY PULSED HYDROGEN-EXCHANGE
KINETICS OF HYDROGEN-BOND BREAKAGE IN THE PROCESS OF UNFOLDING OF RIBONUCLEASE-A MEASURED BY PULSED HYDROGEN-EXCHANGE
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DOI：
10.1073/pnas.92.7.2657
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发表时间：
1995-03-28
影响因子：
11.1
通讯作者：
BALDWIN, RL
中科院分区：
文献类型：
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作者：
KIEFHABER, T;BALDWIN, RL
对核糖核酸酶A(EC 3.1.27.5)的动力学展开中间体进行了灵敏的测试，该酶在缓慢展开的条件下(10℃，pH 8.0，4.5M氯化胍)。用多肽NH质子交换(H-2-H-1)来监测蛋白质去折叠过程中单个氢键的结构开放，并建立了蛋白质去折叠过程中氢交换的动力学模型。分析表明，在此条件下，核糖核酸酶A的EX1交换(受开放速率限制)可以监测其去折叠的动力学过程。在测量的49个质子的展开/交换动力学中，47个质子具有已知的氢键受体基团。为了测试展开过程中的交换是遵循EX2(碱催化)还是EX1(非催化)机制，在pH 8，O和pH 9.0下都测量了展开/交换，发现一些交换较快的质子同时进行EX1和EX2过程交换，但在pH 8时43个交换较慢的质子只通过EX1机制进行交换，它们具有非常相似的速率。因此，很可能所有49个质子都以相同的速率进行EX1交换。结果表明，在展开过程中的单个限速步骤破坏了整个肽氢键网络，并导致核糖核酸酶A的整体展开。观察到的一些遵循EX2机制的质子的额外交换可能是平衡展开中间体的结果，将在其他地方讨论。
A sensitive test for kinetic unfolding intermediates in ribonuclease A (EC 3.1.27.5) is performed under conditions where the enzyme unfolds slowly (10 degrees C, pH 8.0, 4.5 M guanidinium chloride). Exchange of peptide NH protons (H-2-H-1) is used to monitor structural opening of individual hydrogen bonds during unfolding, and kinetic models are developed for hydrogen exchange during the process of protein unfolding. The analysis indicates that the kinetic process of unfolding can be monitored by EX1 exchange (limited by the rate of opening) for ribonuclease A in these conditions. Of the 49 protons whose unfolding/exchange kinetics was measured, 47 have known hydrogen bond acceptor groups. To test whether exchange during unfolding follows the EX2 (base-catalyzed) or the EX1 (uncatalyzed) mechanism, unfolding/exchange was measured both at pH 8.,O and at pH 9.0, A few faster-exchanging protons were found that undergo exchange by both EX1 and EX2 processes, but the 43 slower-exchanging protons at pH 8 undergo exchange only by the EX1 mechanism, and they have closely similar rates. Thus, it is likely that all 49 protons undergo EX1 exchange at the same rate. The results indicate that a single rate-limiting step in unfolding breaks the entire network of peptide hydrogen bonds and causes the overall unfolding of ribonuclease A. The additional exchange observed for some protons that follows the EX2 mechanism probably results from equilibrium unfolding intermediates and will be discussed elsewhere.