Data from: ProtASR: an evolutionary framework for ancestral protein reconstruction with selection on folding stability

The computational reconstruction of ancestral proteins provides information on past biological events and has practical implications for biomedicine and biotechnology. Currently available tools for ancestral sequence reconstruction (ASR) are often based on empirical amino acid substitution models that assume that all sites evolve at the same rate and under the same process. However, this assumption is frequently violated because protein evolution is highly heterogeneous due to different selective constraints among sites. Here, we present ProtASR, a new evolutionary framework to infer ancestral protein sequences accounting for selection on protein stability. First, ProtASR generates site-specific substitution matrices through the structurally constrained mean-field substitution model (MF), which considers both unfolding and misfolding stability. We previously showed that MF models outperform empirical amino acid substitution models, as well as other structurally constrained substitution models, both in terms of likelihood and correctly inferring amino acid distributions across sites. In the second step, ProtASR adapts a well-established maximum-likelihood (ML) ASR procedure to infer ancestral proteins under MF models. A known bias of ML ASR methods is that they tend to overestimate the stability of ancestral proteins by under-estimating the frequency of deleterious mutations. We compared ProtASR under MF to two empirical substitution models (JTT and CAT), reconstructing the ancestral sequences of simulated proteins. ProtASR yields reconstructed proteins with less biased stabilities, which are significantly closer to those of the simulated proteins. Analysis of extant protein families suggests that folding stability evolves through time across protein families, potentially reflecting neutral fluctuation. Some families exhibit a more constant protein folding stability, while others are more variable. ProtASR is freely available from https://github.com/miguelarenas/protasr and includes detailed documentation and ready-to-use examples. It runs in seconds/minutes depending on protein length and alignment size.祖先蛋白质的计算重建可为过往生物学事件提供关键信息，同时在生物医学与生物技术领域具备实际应用价值。当前可用的祖先序列重建（Ancestral Sequence Reconstruction，ASR）工具，大多基于经验性氨基酸替换模型，这类模型假设所有蛋白质位点以相同速率、遵循统一演化过程发生变异。然而，由于蛋白质不同位点受到的选择约束存在差异，其演化过程往往存在高度异质性，这一假设常常无法成立。 本文提出ProtASR——一种全新的演化分析框架，可在推断祖先蛋白质序列时纳入蛋白质稳定性选择的影响。该框架分为两个步骤：首先，ProtASR通过结构约束平均场替换模型（structurally constrained mean-field substitution model，MF）生成位点特异性替换矩阵，该模型同时考虑了蛋白质的解折叠与错配折叠稳定性。我们此前的研究已证实，相较于经验性氨基酸替换模型及其他结构约束型替换模型，MF模型在似然值计算与跨位点氨基酸分布的准确推断两方面均表现更优。其次，ProtASR对成熟的最大似然（maximum-likelihood，ML）祖先序列重建流程进行适配，以基于MF模型推断祖先蛋白质。已知最大似然祖先序列重建方法存在一项固有偏差：其往往会低估有害突变的发生频率，进而高估祖先蛋白质的稳定性。 我们针对模拟蛋白质的祖先序列重建任务，将基于MF模型的ProtASR与两款经验性替换模型（JTT与CAT）进行了对比。结果显示，ProtASR所重建的蛋白质稳定性偏差更低，与模拟蛋白质的真实稳定性更为接近。对现存蛋白质家族的分析表明，不同蛋白质家族的折叠稳定性随时间发生演化，这一现象或可反映中性漂变过程。部分蛋白质家族的折叠稳定性维持相对恒定，而另一些家族的稳定性则波动更为明显。 ProtASR可从https://github.com/miguelarenas/protasr 免费获取，且附带详细文档与可直接运行的示例程序。其运行耗时依蛋白质长度与序列比对规模而定，仅需数秒至数分钟。