Pan Y, Daggett V
Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195-7610, USA.
Biochemistry. 2001 Mar 6;40(9):2723-31. doi: 10.1021/bi0022036.
Previous molecular dynamics (MD) simulations of thermal denaturation of chymotrypsin inhibitor 2 (CI2) have provided transition-state models in good agreement with experiment. Unfortunately, however, the comparisons have been necessarily indirect. The simulations have provided detailed structural information but not energetics, while from experiment, structure is inferred from a ratio of free energy changes upon mutation (Phi values). Here, direct comparison with experimental free energies is obtained by performing free energy perturbation calculations of hydrophobic deletion mutants of CI2 using transition- and denatured-state structures from various denaturation MD simulations. The agreement between the calculated and experimental DeltaDeltaG and Phi values is quite good (R = 0.8-0.9). In addition, given the availability of realistic atomic models for the denatured protein, the common approach of using small peptides to represent the denatured state in stability calculations can now be evaluated. To this end, two different extended tripeptide models were used: one using the sequence from the protein with the residue to be mutated in the center and the other with this residue surrounded by Ala residues. The results for the two peptides agree neither with one another nor with the different full-length denatured-state models, which do provide results in good agreement with experiment. This finding is noteworthy because the denatured state of CI2 is very disrupted with little residual structure, such that the peptides might have been expected to serve as reasonable models. Overall the calculations presented here validate our previous MD-generated transition- and denatured-state models and therefore the simulated unfolding pathways and their relevance to refolding.
先前对胰凝乳蛋白酶抑制剂2（CI2）热变性的分子动力学（MD）模拟提供了与实验结果高度一致的过渡态模型。然而，遗憾的是，这些比较必然是间接的。模拟提供了详细的结构信息，但没有能量学信息，而从实验中，结构是根据突变时自由能变化的比率（Phi值）推断出来的。在这里，通过使用来自各种变性MD模拟的过渡态和变性态结构对CI2的疏水缺失突变体进行自由能扰动计算，获得了与实验自由能的直接比较。计算得到的ΔΔG和Phi值与实验值之间的一致性相当好（R = 0.8 - 0.9）。此外，鉴于变性蛋白有逼真的原子模型，现在可以评估在稳定性计算中使用小肽来代表变性态的常用方法。为此，使用了两种不同的延伸三肽模型：一种使用蛋白质的序列，将待突变的残基置于中心，另一种是该残基被丙氨酸残基包围。这两种肽的结果彼此不一致，也与不同的全长变性态模型不一致，而全长变性态模型确实提供了与实验结果高度一致的结果。这一发现值得注意，因为CI2的变性态非常无序，几乎没有残留结构，因此可能预计这些肽会是合理的模型。总体而言，这里给出的计算验证了我们之前由MD生成的过渡态和变性态模型，从而验证了模拟的展开途径及其与重折叠的相关性。
