Biology subjects

Theobald, D. L.

Publications and source records attributed to Theobald, D. L..

7 recordsLinked to original sources

bioRxiv2026

GTRspmix: Capturing Heterogeneity of Exchangeabilities Across Sites to Improve Protein Phylogenetics

Site rate and profile mixture models capture the heterogeneity of the amino acid substitution process across sites. However, these models typically use a single matrix of amino acid exchangeabilities and ignore potential heterogeneities of these exchangeabilities across sites. Simply combining multiple exchangeability matrices with rate and profile mixtures leads to a combinatorial explosion of mixture components and a prohibitive increase in free parameters. Here, we introduce GTRspmix, a novel framework that incorporates multiple exchangeability matrices into profile and site rate mixture models while effectively managing model complexity. GTRspmix employs a clustering-based strategy that groups profiles and assigns a distinct exchangeability matrix to each profile cluster. Evaluations using both empirical and simulated datasets demonstrate that GTRspmix fits empirical data significantly better than conventional models, and that overparameterization does not present a problem for sufficiently large alignments. Based on these results, we estimated general-purpose empirical models (SXXpfamCYY series available in IQ-TREE3) from the Pfam database. These general-purpose models not only fit data much better, but they also influence branch length and tree topology estimates, effectively mitigating long-branch attraction artifacts. Because the total number of rate matrices remains manageable, the computational efficiency of the inference is identical to that of conventional profile mixture models (e.g., LG+C60+G4). GTRspmix provides a more realistic and flexible model of protein evolution, offering a robust foundation for the inference of reliable phylogenetic trees.

evolutionary biology↗

bioRxiv2026

An activity-resistance tradeoff constrains enzyme evolution

The presence of self-resistance genes in antibiotic-producing organisms poses a paradox: how can resistance evolve before the antibiotic exists, and how can an antibiotic producer arise without first evolving resistance? Here we examine the evolutionary origins of self-resistance to mycophenolic acid (MPA), an inhibitor of inosine monophosphate dehydrogenase (IMPDH). The MPA biosynthetic gene cluster (BGC) includes a resistant IMPDH-B. Homologs of IMPDH-B occur not only in MPA producers but also in many non-producing fungi, where remnants of the MPA BGC remain detectable. The phylogeny of IMPDH-B is incongruent with the fungal species tree, consistent with multiple horizontal gene transfer events between Aspergillus and Sordariomycetes. We characterized eleven extant IMPDH-Bs, five from MPA producers and six from nonproducers, along with seven resurrected ancestral enzymes (Anc1-Anc7). MPA resistance appeared between Anc2 and Anc3 and coincided with a loss of catalytic efficiency. Across both ancestral and extant enzymes, MPA resistance correlated strongly with reduced activity, revealing a robust activity-resistance trade-off that has persisted for millions of years. Unexpectedly, both the IMPDH-Bs and ancestral enzymes Anc3-Anc7 were also resistant to ribavirin-5'-monophosphate (RVP), an IMP-competitive inhibitor. Because MPA and RVP bind to similar enzyme conformations, the activity-resistance trade-off likely reflects a design constraint imposed by the need to maintain resistance to multiple inhibitors. Intriguingly, although Anc1 and Anc2 are equally sensitive to MPA, Anc2 shows reduced susceptibility to RVP. This pattern suggests that pre-existing resistance to another IMPDH inhibitor may have created a permissive background for the later evolution of MPA biosynthesis. SignificanceAntibiotic producers must be resistant to the toxins that they produce, but how such self-resistance develops is a mystery. The mycophenolic acid (MPA) biosynthetic gene cluster (BGC) encodes a resistant variant of the MPA target IMPDH (IMPDH-B). Many fungi retain IMPDH-B although they have lost the ability to produce MPA. The IMPDH-B and species phylogenies are incongruent, suggesting evolution of the BGC was complicated. MPA resistance correlates with low catalytic efficiency in modern and ancestral IMPDHs, revealing a robust design constraint tradeoff. Surprisingly, IMPDH-Bs are also resistant to an IMP-competitive inhibitor (RVP). RVP resistance appears to have emerged before MPA resistance. Perhaps resistance to RVP created a background that permitted the genesis of a new toxin.

evolutionary biology↗

bioRxiv2025

Comparing the evolvability of an ancestrally reconstructed and modern adenylate kinase

Directed evolution transformed protein engineering by providing a customizable framework for generating enzymes with improved catalytic performance across diverse functions. Yet modern enzymes often stall during directed evolution because populations become trapped on local fitness peaks. Researchers have suggested that ancestral enzymes offer better starting points because they are typically more thermostable. Here we propose and experimentally test an alternative explanation that does not depend on ancestral thermostability. We posit that ancestrally reconstructed sequences are unusually evolvable because they are inferred from the evolutionary lineages that survived to produce extant proteins. Less evolvable ancestors, and the trajectories emanating from them, disappeared by extinction and therefore do not contribute to reconstructed ancestors. Using thermophilic ancestral and modern adenylate kinases, we performed independent single-round selection experiments for activity in vivo and in vitro. In both settings, the ancestral enzyme tolerates a larger number of mutations, yielding more viable variants with greater genetic diversity than its modern descendants. Because mutational robustness promotes evolvability, these results support an intrinsic evolvability of reconstructed ancestral sequences that makes them superior launch points for directed evolution.

biochemistry↗

bioRxiv2023

Structure of an insect gustatory receptor

Gustatory Receptors (GRs) are critical for insect chemosensation and are potential targets for controlling pests and disease vectors. However, GR structures have not been experimentally determined. We present structures of Bombyx mori Gr9 (BmGr9), a fructose-gated cation channel, in agonist-free and fructose-bound states. BmGr9 forms a tetramer similar to distantly related insect Olfactory Receptors (ORs). Upon fructose binding, BmGr9s ion channel gate opens through helix S7b movements. In contrast to ORs, BmGR9s ligand-binding pocket, shaped by a kinked helix S4 and a shorter extracellular S3-S4 loop, is larger and solvent accessible in both agonist-free and fructose-bound states. Also unlike ORs, fructose binding by BmGr9 involves helix S5 and a binding pocket lined with aromatic and polar residues. Structure-based sequence alignments reveal distinct patterns of ligand-binding pocket residue conservation in GR subfamilies associated with distinct ligand classes. These data provide insight into the molecular basis of GR ligand specificity and function.

biochemistry↗

bioRxiv2023

Strong epistasis constrains the evolution of strict substrate specificity in apicomplexan lactate dehydrogenases

The homologous enzymes lactate and malate dehydrogenase (L/MDH) are structurally similar but are specific for different substrates. LDH vs MDH specificity is canonically governed by the identity of a single "specificity residue" at position 102. However, LDH function has convergently evolved from a specific MDH at least four times, and the catalytic role of residue 102 is not conserved between different phyla. The apicomplexa are a phylum of obligate, intracellular eukaryotic parasites responsible for wide-spread disease such as Plasmodium falciparum (malaria), Cryptosporidium parvum (cryptosporidiosis), Toxoplasma gondii (toxoplasmosis), and Eimeria maxima (eimeriosis). The apicomplexan LDH evolved via a five-residue insertion that produced a novel specificity residue, W107f. The commonly accepted mechanism of LDH specificity involves charge balance and steric occlusion, but our data shows that the general mechanism of apicomplexan LDHs does not use W107f as a steric block. Only Plasmodium LDHs evolved substantial steric specificity, making them exceptional among Apicomplexa. Strong protein epistasis constrained this evolution, making it difficult to revert to ancestral phenotypes. Here, we use ancestral sequence reconstruction (ASR), steady-state kinetics, and x-ray crystallography to characterize apicomplexan LDHs which challenge current assumptions about the evolution of L/MDH activity. We demonstrate the unique specificity of Plasmodium LDHs and identify the active site residues controlling their substrate recognition. The extraordinarily high specificity of Plasmodium LDHs presents difficulties for small-molecule inhibitor development, and successful drugs against Plasmodium LDH may not be efficacious against other Apicomplexa LDHs and their diseases.

evolutionary biology↗

bioRxiv2023

Stability of Alternative Bacteriorhodopsin Folds

Bacteriorhodopsin is a light-activated proton pump found in archaea and some single-celled eukaryotes (Findlay and Pappin, 1986; Sharma et al., 2006; Spudich et al., 2000). This protein adopts the 7TM fold found used by all type I and type II rhodopsins. In previous work, we used bacteriorhodopsin from Haloterrigena turkmenica to demonstrate that substantially altered protein folds exhibit light-activated proton pumping (Kamo et al., 2006; Mackin et al., 2014). In this work, we further characterized these novel folds by assessing the stability of our mutants. We used SDS denaturation to calculate the change in unfolding free energy relative to the wild-type (Cao et al., 2012). We also determine the extinction coefficient for each mutant. These results demonstrate that even dramatic structural rearrangements do not critically destabilize the protein, although the extinction coefficient does vary independently of the stability and the proton-pumping activity. Interestingly, the position of the A helix in the protein sequence has the largest effect on the stability of the mutant; those mutants where A is not located at a terminus are destabilized compared to the wild-type.

biochemistry↗

bioRxiv2020

A thermodynamic model of protein structure evolution explains empirical amino acid rate matrices

Proteins evolve under a myriad of biophysical selection pressures that collectively control the patterns of amino acid substitutions. Averaged over time and across proteins, these evolutionary pressures are sufficiently consistent to produce global substitution patterns that can be used to successfully find homologues, infer phylogenies, and reconstruct ancestral sequences. Although the factors which govern the variation of protein substitution rates has received much attention, the influence of thermodynamic stability constraints remains unresolved. Here we develop a simple model to calculate amino acid rate matrices from evolutionary dynamics controlled by a fitness function that reports on the thermodynamic effects of amino acid mutations in protein structures. This hybrid biophysical and evolutionary model accounts for nucleotide transition/transversion rate bias, multi-nucleotide codon changes, the number of codons per amino acid, and thermodynamic protein stability. We find that our theoretical model accurately recapitulates the complex pattern of empirical rates observed in common global amino acid substitution matrices used in phylogenetics. These results suggest that selection for thermodynamically stable proteins, coupled with nucleotide mutation bias filtered by the structure of the genetic code, is the primary global driver behind the amino acid substitution patterns observed in proteins throughout the tree of life.

evolutionary biology↗