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揭示过往生物学事件的相关信息，在生物医学与生物技术领域具备重要的实际应用价值。当前可用的祖先序列重建（Ancestral Sequence Reconstruction, ASR）工具通常基于经验性氨基酸替换模型，这类模型假设所有演化位点以统一速率演化且遵循完全一致的演化过程。然而这一假设往往难以成立，由于不同蛋白质位点间存在差异化的选择约束，蛋白质演化过程呈现高度异质性。本文提出ProtASR——一种全新的演化分析框架，可在考虑蛋白质稳定性选择效应的前提下推断祖先蛋白质序列。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，该模型同时考量了蛋白质的解折叠与错配折叠稳定性。我们前期研究已证实，无论是在似然值拟合精度还是位点间氨基酸分布的准确推断方面，平均场模型均优于经验性氨基酸替换模型以及其他结构约束替换模型。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改造，以基于平均场模型完成祖先蛋白质的推断。已知最大似然祖先序列重建方法存在一项固有偏差：由于低估了有害突变的发生频率，这类方法往往会高估祖先蛋白质的稳定性。我们将基于平均场模型的ProtASR与两类经典经验性替换模型（JTT与CAT）进行对比，通过重建模拟蛋白质的祖先序列开展对照实验。实验结果表明，ProtASR所重建的蛋白质稳定性偏差更低，其结果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演化分析显示，不同蛋白质家族的折叠稳定性随时间发生动态变化，这一现象可能反映了中性漂变带来的波动。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呈现更为显著的波动。ProtASR可从https://github.com/miguelarenas/protasr免费获取，且附带详细的官方文档与可直接运行的示例脚本。其运行耗时取决于蛋白质长度与序列联配规模，通常为数秒至数分钟。