HYDROGEN-EXCHANGE IN UNLIGATED AND LIGATED STAPHYLOCOCCAL NUCLEASE
HYDROGEN-EXCHANGE IN UNLIGATED AND LIGATED STAPHYLOCOCCAL NUCLEASE
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DOI：
10.1021/bi00092a011
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发表时间：
1993-10-19
期刊：
影响因子：
2.9
通讯作者：
MARKLEY, JL
中科院分区：
文献类型：
--
作者：
LOH, SN;PREHODA, KE;MARKLEY, JL
用氮-15分辨质子核磁共振波谱法测定了葡萄球菌核酸酶H124L（核酸酶H124L）中超过70%的143个主酰胺氢在其未连接状态和与Ca2+和胸腺嘧啶3',5'-二磷酸的三元配合物中的交换动力学。在37℃和pH* 5.5条件下，对H124L酶中缓慢交换氢的保护因子变化超过一个数量级。这一系列保护因素是在全球和地方结构波动的框架内加以解释的。三个最受保护的氢（K24, L25, M26）映射到中央五链β -桶的第2链。将这些氢暴露于溶剂的开启反应的自由能变化（δ δ度）是根据天然状态和变性状态的交换速率计算的，后者的值是根据模型肽交换研究估计的[Molday， R. S., Englander， S. W., & Kallen， R. G.（1972）生物化学11,150-158]。通过尿素变性实验测得的展开自由能变化值δ tag - op与δ tag - u之间的关系非常接近。因此，这些氢的交换似乎是通过蛋白质的整体展开而发生的。其中一个区域表现出较低的保护因子：它映射到螺旋2和螺旋3的c端部分以及中间部分。该区域被认为是核酸酶的一个次要疏水结构域[Shortle， D., Stites， W. E., & Meeker, a . K. (1990) Biochemistry 29, 8033-8041]。该区域保护因子的降低似乎是由局部结构波动引起的，这种波动伴随着K116-P117肽键的顺式半箭头向右超过半箭头向左的反式异构化。发现抑制剂结合产生保护因子的整体增加。从NH交换数据估计的抑制剂结合所提供的基线稳定性增加（DELTADELTAG-degrees op）再次被发现与从尿素展开实验中确定的DELTADELTAG-degrees u值相似，除了受K116-P117肽键的顺式半箭头右比半箭头左反式异构化影响的区域。该区域显示出额外的保护作用，这是由于抑制剂诱导的顺式半箭头右转平衡比半箭头左转平衡向更稳定的交换顺式构象的扰动。本研究的结果表明，氢交换动力学可以用来估计在配体存在和不存在的情况下H124L核酸酶的整体稳定性，并确定结构自由能的局部变化。此外，从展开状态的汇率似乎很好地描述了随机线圈值。在实验条件下，没有证据表明在变性状态下存在任何稳定的氢键结构，如残基L24， K25或m26所报道的那样。
The exchange kinetics of over 70% of the 143 backbone amide hydrogens in staphylococcal nuclease H124L (nuclease H124L), both in its unligated state and in its ternary complex with Ca2+ and thymidine 3',5'-bisphosphate, have been quantified by nitrogen-15 resolved proton nuclear magnetic resonance spectroscopy. Protection factors for the slowly exchanging hydrogens in unligated nuclease H124L at 37-degrees-C and pH* 5.5 were found to vary by over one order of magnitude. This range of protection factors has been interpreted in the framework of global and local structural fluctuations. The three most highly protected hydrogens (K24, L25, M26) map to strand 2 of the central five-stranded beta-barrel. The free energy change for the opening reaction which exposes these hydrogens to the solvent (DELTAG-degrees op) was calculated from the exchange rates in the native and denatured states, the latter values being estimated from model peptide exchange studies [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158]. Close agreement was found between DELTAG-degrees op and DELTAG-degrees u, the free energy change of unfolding as measured by urea denaturation experiments. Exchange of these hydrogens thus appears to occur via global unfolding of the protein. One region exhibited somewhat lower protection factors: it mapped to the C-terminal portions of helix 2 and helix 3 and to part of the intervening segment. This region has been identified as a minor hydrophobic domain of nuclease [Shortle, D., Stites, W. E., & Meeker, A. K. (1990) Biochemistry 29, 8033-8041]. The decreased protection factors in this region appear to arise from local structural fluctuations that accompany cis half arrow right over half arrow left trans isomerization about the K116-P117 peptide bond. Inhibitor binding was found to produce global increases in protection factors. The baseline stability increase afforded by inhibitor binding estimated from NH exchange data (DELTADELTAG-degrees op) was found again to be similar to the DELTADELTAG-degrees u value determined from urea unfolding experiments except for in the region affected by the cis half arrow right over half arrow left trans isomerization of the K116-P117 peptide bond. This region showed additional protection attributed to inhibitor-induced perturbation of the cis half arrow right over half arrow left trans equilibrium to the more exchange stable cis conformation. The results of the present study demonstrate that hydrogen exchange kinetics can be used to estimate the global stability of nuclease H124L in the presence and absence of ligands and to pinpoint local changes in structural free energy. In addition, the exchange rates from the unfolded state appear to be well described by the random-coil values. The results provide no evidence for the existence of any stable hydrogen-bonded structure in the denatured state-as reported by residues L24, K25, or M26-under the conditions of the experiment.