Reply to Kitahara and Mulder: An ensemble view of protein stability best explains pressure effects in a T4 lysozyme cavity mutant.
Reply to Kitahara and Mulder: An ensemble view of protein stability best explains pressure effects in a T4 lysozyme cavity mutant.
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回复 Kitahara 和 Mulder：蛋白质稳定性的整体观点最好地解释了 T4 溶菌酶空腔突变体中的压力效应。
DOI：
10.1073/pnas.1424002112
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发表时间：
2015
影响因子：
11.1
通讯作者：
Nucci,NathanielV
中科院分区：
文献类型：
--
作者：
Wand,AJoshua;Nucci,NathanielV
Kitahara和Mulder(1)挑战了我们对T4溶菌酶Leu99Ala(L99A)空腔突变的压力微扰研究的解释。Kitahara和Mulder认为，我们错误地放弃了一个模型，即本征状态(NS)与高度结构的高能态(HES)相互转换，以解释在高压下观察到的核磁共振现象。首先要弄清楚“展开”的含义。Nucci等人(2)注意到观察到的反应是“……指示次全球和局部展开事件，并在很大程度上对应于二级结构单元和结构域的协同展开……此外，与北原和穆德所暗示的相反，使用位置分辨的交叉峰强度(积分)是获得NS和未折叠状态系综之间的表观自由能差的有效手段。话虽如此，将北原和穆德的两种结构相互转换模型贬低为部分展开态的系综的原因有很多。蛋白质的压力敏感性是不均匀的，这强烈反对全球两态解释。仅提供Leu133作为与人类基因组学缺乏简单相关性的许多例子的代表。重要的是，Ando等人的小角X射线散射数据(3)表明，压力感生态比唯一结构和紧凑的NS和HES更广泛。因此，通过限制在反胶束中和通过结晶来抑制压力敏感性最符合我们的观点。北原和穆德认为，在Phe-114环填满空穴的情况下，HES的重组缓解了本征状态的压力敏感性。在一项优雅的研究中，Bouvignies等人(4)表明，苯的结合与HES是相互排斥的。因此，苯占据空腔对展开跃迁的抑制与这两种观点都是一致的。所有这些，甚至更多，都被用来支持我们最初的解释。在裁判中5、模拟结果表明，核磁共振谱中的某些效应与两种结构的相互转化是一致的。许多情景都是同样一致的，因此这并不能提供清晰度。他们未能检测到表明高压下高度无序和溶剂化的“随机线圈”共振。虽然很难在Nucci等人的补充数字中辨别出来，但这种共鸣在我们的数据中找到了。这推翻了两国论的解释。他们未能探测到多种状态可能是因为使用了体积很小的核磁共振池，显然没有低温探测器的好处。我们还表明，在达到大于1kbar的压力之前，水不会显著地进入空腔。这与北原和穆尔德直接不一致，那里的HES人继续居住。因此，尽管我们同意这些态的相互转换是在一个使核磁共振谱复杂的时间尺度上进行的，但有压倒性的证据表明，对T4L99A施加压力会导致大量不同的部分展开态的存在，而不是简单地将本征态转移到HES。
Kitahara and Mulder (1) challenge our interpretation of pressure-perturbation studies of the Leu99Ala (L99A) cavity mutant of the T4 lysozyme. Kitahara and Mulder suggest we incorrectly discarded a model where the native state (NS) interconverts with a highly structured high-energy state (HES) to explain NMR phenomena observed at elevated pressure. It is first important to be clear as to the meaning of “unfolding.” Nucci et al.(2) note that the responses observed are “... indicative of subglobal and local unfolding events and largely correspond to the cooperative unfolding of secondary structural units and domains...” Also, contrary to what is implied by Kitahara and Mulder, using siteresolved cross-peak intensities (integrals) is a valid means to obtain the apparent free energy difference between the NS and an ensemble of unfolded states. Having said that, the reasons to discount the Kitahara and Mulder two-structure interconversion model in favor of an ensemble of partially unfolded states are many. The pressure sensitivity of the protein is heterogeneous, which argues strongly against the global two-state interpretation. Leu133 is simply provided as representative of many examples of the lack of simple correlation with the HES. Critically, small angle X-ray scattering data by Ando et al.(3) show that the pressure-induced states are more extensive than the uniquely structured and compact NS and HES. Thus, the suppression of the pressure sensitivity by confinement in a reverse micelle and by crystallization is most consistent with our view. Kitahara and Mulder argue that the pressure sensitivity of the native state is relieved by reorganization to the HES where the ring of Phe-114 fills the cavity. In an elegant study, Bouvignies et al.(4) show that binding of benzene is mutually exclusive with the HES. The suppression of unfolding transitions by occupation of the cavity by benzene is therefore consistent with both views. All of this and more was used to support our original interpretation. In ref. 5, simulations are used to illustrate that some effects in the NMR spectra are consistent with the interconversion of two structures. A multitude of scenarios are equally consistent, so this provides no clarity. They failed to detect “random coil” resonances indicative of high disorder and solvation at high pressure. Although difficult to discern in the supplemental figures to Nucci et al., such resonances are found in our data. This demolishes the two-state interpretation. Their failure to detect the multitude of states may arise from use of a very low volume NMR cell apparently without the benefit of a cryogenic probe. We also show that water does not significantly enter the cavity until pressures greater than 1 kbar are reached. This is directly inconsistent with Kitahara and Mulder, where the HES continues to be populated. Thus, although we agree that interconversion of these states is on a time scale that complicates the NMR spectra, there is overwhelming evidence that the application of pressure to T4 L99A results in the population of a large number of distinct partially unfolded states and does not simply shift the native state to the HES.