ProtASR2: Ancestral Reconstruction of Protein Sequences accounting for Folding Stability

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.