Non-conservation of folding rates in the thioredoxin family reveals degradation of ancestral unassisted-folding

Abstract

Evolution involves not only adaptation, but also the degradation of superfluous features. Many examples of degradation at the morphological level are known (vestigial organs, for instance). However, the impact of degradation on molecular evolution has been rarely addressed. Thioredoxins serve as general oxidoreductases in all cells. Here, we report extensive mutational analyses on the folding of modern and resurrected ancestral bacterial thioredoxins. Contrary to claims from recent literature, in vitro folding rates in the thioredoxin family are not evolutionary conserved, but span at least a ∼100-fold range. Furthermore, modern thioredoxin folding is often substantially slower than ancestral thioredoxin folding. Unassisted folding, as probed in vitro, thus emerges as an ancestral vestigial feature that underwent degradation, plausibly upon the evolutionary emergence of efficient cellular folding-assistance. More generally, our results provide evidence that degradation of ancestral features shapes, not only morphological evolution, but also the evolution of individual proteins.

Crystal structure of chloroplastic thioredoxin z defines a novel type-specific target recognition

“…P134 is built in the cis conformation. As reported earlier, this cis-proline is an important kinetic bottleneck for protein folding [81] and is already observed in inferred precambrian TRXs [82]. Altogether, redox site microenvironment and structural-dependent functional features of TRXs are present and canonical in CrTRXz.…”

Evidence for a role of phenotypic mutations in virus adaptation

“…Some of these molecules will still recruit the original thioredoxin, whereas some other will have the capability to recruit the alternative LPBCA thioredoxin. Note that the putative ancestral protein we have used displays higher stability (Perez-Jimenez et al, 2011) and better folding kinetics properties (Gamiz-Arco et al, 2019) than its modern E. coli counterpart, which should contribute to somewhat higher levels of folded protein in vivo. This explains that lysis times for the engineered host eventually become somewhat smaller than the lysis times for the original host in experiment C (Figure 8B).…”

Combining ancestral reconstruction with folding-landscape simulations to engineer heterologous protein expression

“…In vitro redox activity, as determined by the insulin aggregation assay and by the assay with thioredoxin reductase coupled to DTNB, is also similar for the three thioredoxins (Fig S1). The two modern thioredoxins display similar high stability as shown by midpoint of about 8M for urea denaturation experiments 47 and by denaturation temperatures above 80°C (see details below). The ancestral LPBCA thioredoxin is a hyperstable protein that cannot be denatured by urea at room temperature and that has a denaturation temperature of about 123°C 48,60 .…”