Susceptibility to endosomal degradation is a decisive contribution to a protein's immunogenicity. It is assumed that the processing kinetics of structured proteins are inherently linked to their probability of local unfolding. In this study, we quantify the impact of endosomal acidification on the conformational stability of the major timothy grass pollen allergen Phl p 6. We use state of the art sampling approaches in combination with constant pH MD techniques to profile pH-dependent local unfolding events in atomistic detail. Integrating our findings into the current view on type 1 allergic sensitization, we characterize local protein dynamics in the context of proteolytic degradation at neutral and acidic pH for the wild type protein and point mutants with varying proteolytic stability. We analyze extensive simulation data using Markov state models and retrieve highly reliable thermodynamic and kinetic information at varying pH levels. Thereby we capture the impact of endolysosomal acidification on the structure and dynamics of the Phl p 6 mutants. We find that upon protonation at lower pH values, the conformational flexibilities in key areas of the wild type protein, i.e., T-cell epitopes and early proteolytic cleavage sites, increase significantly. A decrease of the pH even leads to local unfolding in otherwise stable secondary structure elements, which is a prerequisite for proteolytic cleavage. This effect is even more pronounced in the destabilized mutant, while no unfolding was observed for the stabilized mutant. In summary, we report detailed structural models which rationalize the experimentally observed cleavage pattern during endosomal acidification.

对内体降解的敏感性是对蛋白质免疫原性的决定性贡献。据推测，结构化蛋白质的加工动力学与其局部展开的概率有着内在的联系。在这项研究中，我们量化了内体酸化对主要梯牧草花粉过敏原 Phl p 6 构象稳定性的影响。我们使用最先进的采样方法结合恒定 pH MD 技术来分析 pH 依赖性局部展开事件原子细节。将我们的研究结果整合到当前关于 1 型过敏性致敏的观点中，我们描述了在中性和酸性 pH 条件下蛋白水解降解背景下野生型蛋白质和具有不同蛋白水解稳定性的点突变体的局部蛋白质动力学特征。我们使用马尔可夫状态模型分析大量模拟数据，并在不同的 pH 水平下检索高度可靠的热力学和动力学信息。因此，我们捕获了内溶酶体酸化对 Phl p 6 突变体的结构和动力学的影响。我们发现在较低 pH 值下质子化后，野生型蛋白质关键区域（即 T 细胞表位和早期蛋白水解切割位点）的构象灵活性显着增加。pH 值的降低甚至会导致其他稳定的二级结构元素的局部展开，这是蛋白水解切割的先决条件。这种效应在不稳定的突变体中更为明显，而在稳定的突变体中没有观察到展开。总之，