DeMarco Mari L, Alonso Darwin O V, Daggett Valerie
Biomolecular Structure and Design Program, University of Washington, Seattle 98195-7610, USA.
J Mol Biol. 2004 Aug 20;341(4):1109-24. doi: 10.1016/j.jmb.2004.06.074.
Proteins with ultra-fast folding/unfolding kinetics are excellent candidates for study by molecular dynamics. Here, we describe such simulations of a three helix bundle protein, the engrailed homeodomain (En-HD), which folds via the diffusion-collision model. The unfolding pathway of En-HD was characterized by seven simulations of the protein and 12 simulations of its helical fragments yielding over 1.1 micros of simulation time in water. Various conformational states along the unfolding pathway were identified. There is the compact native-like transition state, a U-shaped helical intermediate and an unfolded state with dynamic helical segments. Each of these states is in good agreement with experimental data. Examining these states as well as the transitions between them, we find the role of long-range tertiary contacts, specifically salt-bridges, important in the folding/unfolding pathway. In the folding direction, charged residues form long-range tertiary contacts before the hydrophobic core is formed. The formation of HII is assisted by a specific salt-bridge and by non-specific (fluctuating) tertiary contacts, which we call contact-assisted helix formation. Salt-bridges persist as the protein approaches the transition state, stabilizing HII until the hydrophobic core is formed. To complement this information, simulations of fragments of En-HD illustrate the helical propensities of the individual segments. By thermal denaturation, HII proved to be the least stable helix, unfolding in less than 450 ps at high temperature. We observed the low helical propensity of C-terminal residues from HIII in fragment simulations which, when compared to En-HD unfolding simulations, link the unraveling of HIII to the initial event that drives the unfolding of En-HD.
具有超快折叠/去折叠动力学的蛋白质是通过分子动力学进行研究的极佳候选对象。在此，我们描述了对一种三螺旋束蛋白—— engrailed 同源结构域（En-HD）的此类模拟，该蛋白通过扩散碰撞模型进行折叠。通过对该蛋白进行七次模拟以及对其螺旋片段进行十二次模拟，在水中产生了超过 1.1 微秒的模拟时间，从而对 En-HD 的去折叠途径进行了表征。确定了去折叠途径上的各种构象状态。存在紧凑的类似天然态的过渡态、U 形螺旋中间体以及具有动态螺旋片段的未折叠态。这些状态中的每一种都与实验数据高度吻合。通过研究这些状态以及它们之间的转变，我们发现远程三级相互作用，特别是盐桥，在折叠/去折叠途径中起着重要作用。在折叠方向上，带电残基在疏水核心形成之前就形成了远程三级相互作用。特定盐桥和非特异性（波动的）三级相互作用辅助了 HII 的形成，我们将其称为接触辅助螺旋形成。当蛋白质接近过渡态时，盐桥持续存在，稳定 HII 直至疏水核心形成。为补充这些信息，对 En-HD 片段的模拟阐明了各个片段的螺旋倾向。通过热变性实验，证明 HII 是最不稳定的螺旋，在高温下不到 450 皮秒就会去折叠。我们在片段模拟中观察到 HIII 的 C 末端残基螺旋倾向较低，与 En-HD 去折叠模拟相比，这将 HIII 的解旋与驱动 En-HD 去折叠的初始事件联系起来。
