pH-Dependent conformational changes in proteins and their effect on experimental pK(a)s: the case of Nitrophorin 4.
pH-Dependent conformational changes in proteins and their effect on experimental pK(a)s: the case of Nitrophorin 4.
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DOI：
10.1371/journal.pcbi.1002761
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发表时间：
2012
影响因子：
4.3
通讯作者：
Roitberg AE
中科院分区：
文献类型：
--
作者：
Di Russo NV;Estrin DA;Martí MA;Roitberg AE
由于氨基酸在酶催化、底物结合和蛋白质结构中具有重要作用，其酸碱行为是一个重要的研究课题。由于与蛋白质环境的相互作用，它们的pKa值可能会偏离其在溶液中的数值，并且如果一种蛋白质具有两种稳定的构象，一个残基可能具有不同的、取决于构象的“微观”pKa值。在这些情况下，由于pH、质子化状态和蛋白质构象之间的耦合，对pKa的实验测量结果的解释变得复杂。我们利用硝基铁载体蛋白4（NP4）研究了这些问题，这种蛋白质以pH敏感的方式释放一氧化氮。在pH 5.5时，NP4处于关闭构象，一氧化氮紧密结合；而在pH 7.5时，天冬氨酸30（Asp30）去质子化，导致构象变为开放状态，一氧化氮可轻易逸出。通过恒pH分子动力学研究，我们发现了两个不同的Asp30微观pKa值：在关闭结构中为8.5，在开放结构中为4.3。然后，利用一个四态模型，我们将所得到的微观值与实验观测到的“表观”pKa值相关联，得到的值为6.5，与实验数据高度吻合。这个值必须被解释为从关闭态到开放态的群体转变发生时的pH值。更普遍地说，我们的结果表明，可以将微观的、取决于结构的pKa值与实验观测到的、取决于集合的表观pKa值相关联，并且在NP4这个相对简单的案例中所获得的见解在一些涉及pH依赖性转变的更复杂案例中可能是有用的，这些案例具有重要的生化意义。
氨基酸与其蛋白质环境的相互作用可能导致一种酸碱行为，这种行为与在溶液中观察到的行为有很大不同。当蛋白质改变构象时，这种环境可能会发生很大变化。因此，氨基酸将具有两个不同的“微观”pKa值。硝基铁载体蛋白4是研究这种行为的一个很好的案例，因为它经历了一种pH依赖性的构象变化，这种变化在实验上得到了很好的表征。利用计算机模拟工具，我们发现关键的可滴定天冬氨酸30具有两个非常不同的微观pKa值：4.3和8.5，这与在溶液中观察到的转变pKa值有显著差异。然而，利用一个简单的模型，我们能够理解这是如何导致在实验测量的pH约为6.5时发生构象变化的。在这个相对简单的案例中获得的见解在其他更复杂的案例中可能是有用的，在这些案例中，表观pKa也是不同构象相互作用的结果，其中一些氨基酸处于非常不同的环境中。
The acid-base behavior of amino acids is an important subject of study due to their prominent role in enzyme catalysis, substrate binding and protein structure. Due to interactions with the protein environment, their pKas can be shifted from their solution values and, if a protein has two stable conformations, it is possible for a residue to have different “microscopic”, conformation-dependent pKa values. In those cases, interpretation of experimental measurements of the pKa is complicated by the coupling between pH, protonation state and protein conformation. We explored these issues using Nitrophorin 4 (NP4), a protein that releases NO in a pH sensitive manner. At pH 5.5 NP4 is in a closed conformation where NO is tightly bound, while at pH 7.5 Asp30 becomes deprotonated, causing the conformation to change to an open state from which NO can easily escape. Using constant pH molecular dynamics we found two distinct microscopic Asp30 pKas: 8.5 in the closed structure and 4.3 in the open structure. Using a four-state model, we then related the obtained microscopic values to the experimentally observed “apparent” pKa, obtaining a value of 6.5, in excellent agreement with experimental data. This value must be interpreted as the pH at which the closed to open population transition takes place. More generally, our results show that it is possible to relate microscopic structure dependent pKa values to experimentally observed ensemble dependent apparent pKas and that the insight gained in the relatively simple case of NP4 can be useful in several more complex cases involving a pH dependent transition, of great biochemical interest. The interaction of an amino acid with its protein environment can result in an acid-base behavior that is very different from what would be observed in solution. This environment can be greatly altered when the protein changes conformation. As a result, the amino acid will have two different “microscopic” pKa values. Nitrophorin 4 is a good case study to explore this behavior, because it undergoes a pH-dependent conformational change that is well characterized experimentally. Using computer simulation tools, we found that the key titratable Aspartic acid 30, has two very different microscopic pKas: 4.3 and 8.5, which are significantly different to the observed transition pKa in solution. However, using a simple model, we were able to understand how this causes the conformational change to take place at pH∼6.5, as measured experimentally. The insight gained in this relatively simple case can be useful in other more complex cases where the apparent pKa is also a result of the interplay of different conformations where some amino acids experience very different environments.
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影响因子：
2.9
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通讯作者：
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