Li A, Daggett V
Department of Medicinal Chemistry, University of Washington, Seattle 98195-7610, USA.
J Mol Biol. 1998 Jan 30;275(4):677-94. doi: 10.1006/jmbi.1997.1484.
The folding/unfolding pathway of barnase has been studied extensively using the protein engineering method, which has provided indirect structural information for the transition state and the major folding intermediate. To further characterize the structural properties of the intermediate, we have simulated the thermal denaturation of barnase beginning from the average NMR structure. Our results indicate that there are at least two intermediates on the unfolding pathway. The three hydrophobic cores are partially formed in the major intermediate (I1), with core1 and core3 being slightly stronger than core2. Helix alpha 1 is substantially formed, with the center being stronger than the termini. The first turn of alpha 2 is lost and alpha 3 is unfolded. The center of the beta-sheet is substantially formed, but the edges are disrupted. These structural characteristics are in good qualitative agreement with the experimental data. For semi-quantitative comparison with experimental data, the extent of native structure of individual residues is characterized by a structure index, S, that reflects both secondary and tertiary structure. There is good agreement between S and the experimentally measured phi values, which are based on energetics, except for three residues. These residues are polar and non-conservative mutations were made to obtain phi values, which can complicate structural interpretations. These residues make strong side-chain interactions in I1, but the backbone structure is disrupted, leading to low S values. Thus, this discordance highlights possible limitations in both the phi value and S value analyses: strong polar interactions in the intermediate may give rise to high phi values that are not reflective of structure per se; however, due to sampling limitations, any one simulation is not expected to capture all of the features of the true conformational ensemble. In any case, these simulations provide an experimentally testable, atomic-level structural model for the major folding intermediate of barnase, as well as the detailed pathway from the native to the intermediate state.
利用蛋白质工程方法对核糖核酸酶 barnase 的折叠/去折叠途径进行了广泛研究，该方法为过渡态和主要折叠中间体提供了间接的结构信息。为了进一步表征中间体的结构特性，我们从平均核磁共振结构开始模拟了 barnase 的热变性。我们的结果表明，在去折叠途径上至少存在两个中间体。在主要中间体（I1）中，三个疏水核心部分形成，核心 1 和核心 3 比核心 2 稍强。α1 螺旋基本形成，中间部分比末端更强。α2 的第一圈丢失，α3 去折叠。β 折叠的中心基本形成，但边缘被破坏。这些结构特征与实验数据在定性上吻合良好。为了与实验数据进行半定量比较，单个残基的天然结构程度用结构指数 S 来表征，S 反映了二级和三级结构。除了三个残基外，S 与基于能量学的实验测量的 φ 值之间有很好的一致性。这些残基是极性的，并且进行了非保守突变以获得 φ 值，这可能会使结构解释复杂化。这些残基在 I1 中形成了很强的侧链相互作用，但主链结构被破坏，导致 S 值较低。因此，这种不一致突出了 φ 值和 S 值分析中可能存在的局限性：中间体中的强极性相互作用可能导致高 φ 值，而这些值并不能反映结构本身；然而，由于采样限制，任何一个模拟都不太可能捕捉到真实构象集合的所有特征。无论如何，这些模拟为 barnase 的主要折叠中间体以及从天然态到中间体状态的详细途径提供了一个可通过实验检验的原子水平结构模型。
