DOI: 10.5061/dryad.vdncjsxqd
title: ProtASR2: Ancestral Reconstruction of Protein Sequences accounting for Folding Stability

description: 1. The ancestral sequence reconstruction (ASR) is a molecular evolution
 technique that provides applications to a variety of fields such as
 biotechnology and biomedicine. In order to infer ancestral sequences with
 realistic biological properties, the accuracy of ASR methods is crucial.
 We previously developed an ASR framework for proteins, called ProtASR,
 which is based on our site-specific stability constrained substitution
 (SCS) model with selection on protein folding stability against both
 unfolding and misfolding. This model improved the empirical substitution
 models traditionally applied in ASR without increasing the computational
 complexity. However, it adopted a global exchangeability matrix, an
 approximation that we overcome here by considering site-specific
 exchangeability matrices based on the Halpern-Bruno approach. 2. Here we
 present ProtASR2, a new version of our ASR framework that implements novel
 SCS models of protein evolution, namely mean-field (MF) and wild-type
 (WT). 3. ProtASR2 under MF and WT SCS models outperforms empirical models
 and previous SCS models in terms of goodness of fit and site-specific
 distributions of amino acids. Importantly, the framework infers ancestral
 sequences with more realistic predicted folding stability with respect to
 simulated sequences, while empirical, CAT and other SCS models tend to
 overestimate the folding stability. We applied ProtASR2 to explore the
 evolution of two protein families present in diverse Prokaryota and found
 fluctuations of protein stability over time in both families. ProtASR2 is
 available from https://github.com/miguelarenas/protasr and the new SCS
 models are also available from https://github.com/ugobas/protevol. 4. Use
 of ProtASR2 will allow more realistic inferences of ancestral proteins in
 terms of folding stability with respect to those based on traditional
 empirical and CAT substitution models of protein evolution.
description: Data Availability<br> Raw data (real (downloaded from the
 Pfam database), simulated (with the program ProteinEvolver) and inferred
 protein sequences (with the program ProtASR2) and their corresponding
 protein folding energies (predicted with the program
 <i>DeltaGREM</i>)) that was obtained in this study.
description: <strong>Evaluation of the ASR</strong><br> For
 each protein family, the folder “*SimulatedANDinferredDATA” includes all
 simulated protein sequence alignments (sequences#.fas) and inferred
 ancestral sequences with ProtASR2 under MF and WT models.<br> The
 folder “*ComputedEnergiesFromData” includes the energies of sequences of
 every dataset (files *.dat) presented in the corresponding folder
 “*SimulatedANDinferredDATA”. <strong>Real Data
 used for the ASR</strong><br> Files related with the analysis
 of real data are shown in the folder “RealDataASR”, for the studied
 protein families (DDL and DNAK). MSA and phylogenetic trees are in
 .fas/.nex formats and Newick format, respectively. Energies are shown in
 the .dat file and printed on the tree in files *Energies.tre (we recommend
 open this tree with the program FrigTree). Some derived figures are also
 included. Results are shown for both structurally constrained substitution
 models, MF and WT.