Highly Anomalous Energetics of Protein Cold Denaturation Linked to Folding-Unfolding Kinetics

Despite several careful experimental analyses, it is not yet clear whether protein cold-denaturation is just a “mirror image” of heat denaturation or whether it shows unique structural and energetic features. Here we report that, for a well-characterized small protein, heat denaturation and cold denaturation show dramatically different experimental energetic patterns. Specifically, while heat denaturation is endothermic, the cold transition (studied in the folding direction) occurs with negligible heat effect, in a manner seemingly akin to a gradual, second-order-like transition. We show that this highly anomalous energetics is actually an apparent effect associated to a large folding/unfolding free energy barrier and that it ultimately reflects kinetic stability, a naturally-selected trait in many protein systems. Kinetics thus emerges as an important factor linked to differential features of cold denaturation. We speculate that kinetic stabilization against cold denaturation may play a role in cold adaptation of psychrophilic organisms. Furthermore, we suggest that folding-unfolding kinetics should be taken into account when analyzing in vitro cold-denaturation experiments, in particular those carried out in the absence of destabilizing conditions

Representative examples of fluorescence intensity versus time profiles for thioredoxin unfolding.

<p>The numbers alongside the profiles stand for the temperature and the guanidine concentration used. Experimental data are shown in red and the black line represents the best fit of a single-exponential.</p

Heat and cold denaturation of thioredoxin at pH 7 in the presence of 2M guanidine, as followed by, a) far-UV circular dichroism (222 nm), b) near-UV circular dichroism (280 nm) and, c) differential scanning calorimetry.

<p>The three experiments (far-UV, near-UV and DSC) were carried out with the same solution and following exactly the same heating protocol (see text and “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023050#s3" target="_blank">Materials and Methods</a>” for details). The native-state (N) and unfolded-state (U) profiles in panels a and b were obtained in the absence of guanidine and in the presence of 6M guanidine, respectively.</p

Theoretical simulation of temperature-scanning profiles for protein heat-cold denaturation including slow folding-unfolding kinetics.

<p>a) Folding-unfolding energetics used in the simulation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023050#s3" target="_blank">Materials and Methods</a> for details). Profiles of unfolding free energy and unfolding enthalpy versus temperature are shown. b) Plots of fraction of native protein versus temperature for several values of the temperature scanning-rate. c) Simulated scanning calorimetry profiles at the indicated scan rates. The equilibrium profile (formally corresponding to the limit of zero scan rate) is labeled EQ. Vertical dashed lines crossing the three panels indicate the values of the equilibrium heat- and cold-denaturation temperatures at the enthalpy inversion temperature.</p

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

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 evolutionarily 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. © 2019 The Author(s).This research was supported by FEDER Funds, grant BIO2015-66426-R from the Spanish Ministry of Economy and Competitiveness ( J.M.S.-R.), grant RGP0041/2017 from the Human Frontier Science Program ( J.M.S.-R. and E.A.G.) and National Institutes of Health 1R01AR069137 (E.A.G.), Department of Defence MURI W911NF-16-1-0372 (E.A.G.

Single-molecule paleoenzymology probes the chemistry of resurrected enzymes

A journey back in time is possible at the molecular level by reconstructing proteins from extinct organisms. Here we report the reconstruction, based on sequence predicted by phylogenetic analysis, of seven Precambrian thioredoxin enzymes (Trx), dating back between ~1.4 and ~4 billion years (Gyr). The reconstructed enzymes are up to 32° C more stable than modern enzymes and the oldest show significantly higher activity than extant ones at pH 5. We probed their mechanisms of reduction using single-molecule force spectroscopy. From the force-dependency of the rate of reduction of an engineered substrate, we conclude that ancient Trxs utilize chemical mechanisms of reduction similar to those of modern enzymes. While Trx enzymes have maintained their reductase chemistry unchanged, they have adapted over a 4 Gyr time span to the changes in temperature and ocean acidity that characterize the evolution of the global environment from ancient to modern Earth