Characterization of residual structure in the thermally denatured state of barnase by simulation and experiment: Description of the folding pathway

Residual structure in the denatured state of a protein may contain clues about the early events in folding. We have simulated by molecular dynamics the denatured state of barnase, which has been studied by NMR spectroscopy. An ensemble of 10(4) structures was generated after 2 ns of unfolding and following for a further 2 ns. The ensemble was heterogeneous, but there was nonrandom, residual structure with persistent interactions. Helical structure in the C-terminal portion of helix α1 (residues 13–17) and in helix α2 as well as a turn and nonnative hydrophobic clustering between β3 and β4 were observed, consistent with NMR data. In addition, there were tertiary contacts between residues in α1 and the C-terminal portion of the β-sheet. The simulated structures allow the rudimentary NMR data to be fleshed out. The consistency between simulation and experiment inspires confidence in the methods. A description of the folding pathway of barnase from the denatured to the native state can be constructed by combining the simulation with experimental data from φ value analysis and NMR.

蛋白质变性态中的残留结构，或可为蛋白质折叠的早期事件提供关键线索。我们采用分子动力学（molecular dynamics）模拟了巴纳酶（barnase）的变性态，该蛋白的变性态此前已通过核磁共振波谱法（NMR spectroscopy）得到研究。在2纳秒的解折叠过程及后续2纳秒的模拟后，我们生成了包含10⁴个结构的集合。该结构集合呈现异质性，但仍存在非随机的残留结构与持续稳定的相互作用。我们观察到α螺旋1（α1）的C端区域（残基13–17）与α螺旋2（α2）中存在螺旋结构，同时在β折叠3（β3）与β折叠4（β4）之间存在转角及非天然疏水聚集现象，该结果与核磁共振波谱数据一致。此外，α1中的残基与β折叠的C端区域之间存在三级相互作用。模拟得到的结构可使初步的核磁共振数据得到完善补充。模拟与实验结果的一致性，为该模拟方法提供了可信度支撑。结合本模拟与φ值分析（φ value analysis）、核磁共振波谱的实验数据，可构建出巴纳酶从变性态到天然态的完整折叠通路描述。