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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).)

In order to maintain enzyme stability and activity, chloroplasts use two systems of thiol-disulfide reductases for the control of redox-dependent properties of proteins. Previous studies have revealed that plastid-localized thioredoxins (TRX) and the NADPH-dependent thioredoxin reductase C (NTRC) are important for the reduction of cysteine residues of enzymes involved in chlorophyll synthesis. Very recently, it was shown that the pale green phenotype of the ntrc mutant is suppressed when the contents of 2-cysteine peroxiredoxins (2CP) A and B are decreased. Here, we show that suppression of the ntrc phenotype results from a recovery of wild-type-like redox control of chlorophyll biosynthesis enzymes in ntrc/2cp mutants. The presented results support the conclusion that TRXs rather than NTRC are the predominant reductases mediating the redox-regulation of these enzymes., (© 2018 Federation of European Biochemical Societies.)

Thiol-dependent redox regulation of enzyme activity plays a central role in the rapid acclimation of chloroplast metabolism to ever-fluctuating light availability. This regulatory mechanism relies on ferredoxin reduced by the photosynthetic electron transport chain, which fuels reducing power to thioredoxins (Trxs) via a ferredoxin-dependent Trx reductase. In addition, chloroplasts harbor an NADPH-dependent Trx reductase, which has a joint Trx domain at the carboxyl terminus, termed NTRC. Thus, a relevant issue concerning chloroplast function is to establish the relationship between these two redox systems and its impact on plant development. To address this issue, we generated Arabidopsis ( Arabidopsis thaliana ) mutants combining the deficiency of NTRC with those of Trxs f , which participate in metabolic redox regulation, and that of Trx x , which has antioxidant function. The ntrc-trxf1f2 and, to a lower extent, ntrc-trxx mutants showed severe growth-retarded phenotypes, decreased photosynthesis performance, and almost abolished light-dependent reduction of fructose-1,6-bisphosphatase. Moreover, the combined deficiency of both redox systems provokes aberrant chloroplast ultrastructure. Remarkably, both the ntrc-trxf1f2 and ntrc-trxx mutants showed high mortality at the seedling stage, which was overcome by the addition of an exogenous carbon source. Based on these results, we propose that NTRC plays a pivotal role in chloroplast redox regulation, being necessary for the activity of diverse Trxs with unrelated functions. The interaction between the two thiol redox systems is indispensable to sustain photosynthesis performed by cotyledons chloroplasts, which is essential for early plant development., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Proviral factors are host proteins hijacked by viruses for processes essential for virus propagation such as cellular entry and replication. Pathogens and their hosts co-evolve. It follows that replacing a proviral factor with a functional ancestral form of the same protein could prevent viral propagation without fatally compromising organismal fitness. Here, we provide proof of concept of this notion. Thioredoxins serve as general oxidoreductases in all known cells. We report that several laboratory resurrections of Precambrian thioredoxins display substantial levels of functionality within Escherichia coli. Unlike E. coli thioredoxin, however, these ancestral thioredoxins are not efficiently recruited by the bacteriophage T7 for its replisome and therefore prevent phage propagation in E. coli. These results suggest an approach to the engineering of virus resistance. Diseases caused by viruses may have a devastating effect in agriculture. We discuss how the suggested approach could be applied to the engineering of plant virus resistance., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The relationship between the denaturation temperatures of proteins (Tm values) and the living temperatures of their host organisms (environmental temperatures: TENV values) is poorly understood. Since different proteins in the same organism may show widely different Tm's, no simple universal relationship between Tm and TENV should hold, other than Tm≥TENV. Yet, when analyzing a set of homologous proteins from different hosts, Tm's are oftentimes found to correlate with TENV's but this correlation is shifted upward on the Tm axis. Supporting this trend, we recently reported Tm's for resurrected Precambrian thioredoxins that mirror a proposed environmental cooling over long geological time, while remaining a shocking ~50°C above the proposed ancestral ocean temperatures. Here, we show that natural selection for protein kinetic stability (denaturation rate) can produce a Tm↔TENV correlation with a large upward shift in Tm. A model for protein stability evolution suggests a link between the Tm shift and the in vivo lifetime of a protein and, more specifically, allows us to estimate ancestral environmental temperatures from experimental denaturation rates for resurrected Precambrian thioredoxins. The TENV values thus obtained match the proposed ancestral ocean cooling, support comparatively high Archaean temperatures, and are consistent with a recent proposal for the environmental temperature (above 75°C) that hosted the last universal common ancestor. More generally, this work provides a framework for understanding how features of protein stability reflect the environmental temperatures of the host organisms.

In addition to the standard NADPH thioredoxin reductases (NTRs), plants hold a plastidic NTR (NTRC), with a thioredoxin module fused at the C-terminus. NTRC is an efficient reductant of 2-Cys peroxiredoxins (2-Cys Prxs). The interaction of NTRC and chloroplastic thioredoxin x with 2-Cys Prxs has been confirmed in vivo, by bimolecular fluorescence complementation (BiFC) assays, and in vitro, by isothermal titration calorimetry (ITC) experiments. In comparison with thioredoxin x, NTRC interacts with 2-Cys Prx with higher affinity, both the thioredoxin and NTR domains of NTRC contributing significantly to this interaction, as demonstrated by using the NTR and thioredoxin modules of the enzyme expressed separately. The presence of the thioredoxin domain seems to prevent the interaction of NTRC with thioredoxin x., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Little is known about the evolution of protein structures and the degree of protein structure conservation over planetary time scales. Here, we report the X-ray crystal structures of seven laboratory resurrections of Precambrian thioredoxins dating up to approximately four billion years ago. Despite considerable sequence differences compared with extant enzymes, the ancestral proteins display the canonical thioredoxin fold, whereas only small structural changes have occurred over four billion years. This remarkable degree of structure conservation since a time near the last common ancestor of life supports a punctuated-equilibrium model of structure evolution in which the generation of new folds occurs over comparatively short periods and is followed by long periods of structural stasis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Full or partial burial of ionizable groups in the hydrophobic interior of proteins underlies the large modulation in group properties (modified pK value, high nucleophilicity, enhanced capability of interaction with chemical moieties of the substrate, etc.) linked to biological function. Indeed, the few internal ionizable residues found in proteins are known to play important functional roles in catalysis and, in general, in energy transduction processes. However, ionizable-group burial is expected to be seriously disruptive and, it is important to note, most functional sites contain not just one, but several ionizable residues. Hence, the adaptations involved in the development of function in proteins (through in vitro engineering or during the course of natural evolution) are not fully understood. Here, we explore experimentally how proteins respond to the accumulation of hydrophobic-to-ionizable residue substitutions. For this purpose, we have constructed a combinatorial library targeting a hydrophobic cluster in a consensus-engineered, stabilized form of a small model protein. Contrary to naïve expectation, half of the variants randomly selected from the library are soluble, folded, and active, despite including up to four mutations. Furthermore, for these variants, the dependence of stability with the number of mutations is not synergistic and catastrophic, but smooth and approximately linear. Clearly, stabilized protein scaffolds may be robust enough to withstand many disruptive hydrophobic-to-ionizable residue mutations, even when they are introduced in the same region of the structure. These results should be relevant for protein engineering and may have implications for the understanding of the early evolution of enzymes., (Copyright © 2011 Wiley Periodicals, Inc.)

It is possible to travel back in time 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 markedly higher activity than extant ones at pH 5. We probed the mechanisms of reduction of these enzymes using single-molecule force spectroscopy. From the force dependency of the rate of reduction of an engineered substrate, we conclude that ancient Trxs use chemical mechanisms of reduction similar to those of modern enzymes. Although Trx enzymes have maintained their reductase chemistry unchanged, they have adapted over 4 Gyr to the changes in temperature and ocean acidity that characterize the evolution of the global environment from ancient to modern Earth.

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.

We introduce proteolytic scanning calorimetry, a modification of the differential scanning calorimetry approach to the determination of protein stability in which a proteolytic enzyme (thermolysin) is used to mimic a harsh environment. This methodology allows the straightforward calculation of the rate of irreversible denaturation as a function of temperature and concentration of proteolytic enzyme and, as a result, has the potential to probe efficiently the fundamental biophysical features of protein kinetic stability. In the particular case of Escherichia coli thioredoxin (used as an illustrative example in this article), we find that the rate of irreversible denaturation is determined by 1), the global unfolding mechanism at low thermolysin concentrations, indicating that thermodynamic stability may contribute directly to the kinetic stability of thioredoxin under moderately harsh conditions and 2), the rate of unfolding at high thermolysin concentrations, indicating that the free-energy barrier for unfolding may act as a safety mechanism that ensures significant kinetic stability, even in very harsh environments. This thioredoxin picture, however, is by no means expected to be general and different proteins may show different patterns of kinetic stabilization. Proteolytic scanning calorimetry is particularly well-suited to probe this diversity at a fundamental biophysical level., (Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

A large collection of structural snapshots along a full catalytic cycle of Escherichia coli thioredoxin reductase (TrxR) has been generated and characterized using a combination of theoretical methods. Molecular models were built starting from the available X-ray crystallographic structures of dimeric wild-type TrxR in the flavin-oxidizing conformation and a C135S TrxR mutant enzyme in a flavin-reducing conformation "trapped" by a cross-link between Cys138 of TrxR and Cys32 of C35S mutant thioredoxin (Trx). The transition between these two extreme states, which is shown to be reproduced in a normal mode analysis, as well as natural cofactor binding and dissociation, were simulated for the wild-type species using unrestrained and targeted molecular dynamics following docking of oxidized Trx to reduced TrxR. The whole set of simulations provides a comprehensive structural framework for understanding the mechanism of disulfide reduction in atomic detail and identifying the most likely intermediates that facilitate entry of NADPH and exit of NADP(+). The crucial role assigned to Arg73 and Lys36 of Trx in substrate binding and complex stabilization was ascertained when R73G, R73D, and K36A site-directed mutants of Trx were shown to be impaired to different extents in their ability to be reduced by TrxR. On the basis of previous findings and the results reported herein, E. coli TrxR appears as a beautifully engineered molecular machine that is capable of synchronizing cofactor capture and ejection with substrate binding and redox activity through an interdomain twisting motion., ((c) 2009 Wiley-Liss, Inc.)

Thioredoxins (Trxs) are oxidoreductase enzymes, present in all organisms, that catalyze the reduction of disulfide bonds in proteins. By applying a calibrated force to a substrate disulfide, the chemical mechanisms of Trx catalysis can be examined in detail at the single-molecule level. Here we use single-molecule force-clamp spectroscopy to explore the chemical evolution of Trx catalysis by probing the chemistry of eight different Trx enzymes. All Trxs show a characteristic Michaelis-Menten mechanism that is detected when the disulfide bond is stretched at low forces, but at high forces, two different chemical behaviors distinguish bacterial-origin from eukaryotic-origin Trxs. Eukaryotic-origin Trxs reduce disulfide bonds through a single-electron transfer reaction (SET), whereas bacterial-origin Trxs show both nucleophilic substitution (S(N)2) and SET reactions. A computational analysis of Trx structures identifies the evolution of the binding groove as an important factor controlling the chemistry of Trx catalysis.

Understanding how the catalytic mechanisms of enzymes are optimized through evolution remains a major challenge in molecular biology. The concept of co-evolution implicates that compensatory mutations occur to preserve the structure and function of proteins. We have combined statistical analysis of protein sequences with the sensitivity of single molecule force-clamp spectroscopy to probe how catalysis is affected by structurally distant correlated mutations in Escherichia coli thioredoxin. Our findings show that evolutionary anti-correlated mutations have an inhibitory effect on enzyme catalysis, whereas positively correlated mutations rescue the catalytic activity. We interpret these results in terms of an evolutionary tuning of both the enzyme-substrate binding process and the chemistry of the active site. Our results constitute a direct observation of distant residue co-evolution in enzyme catalysis.

Force-clamp spectroscopy is a novel technique for studying mechanochemistry at the single-bond level. Single disulfide bond reduction events are accurately detected as stepwise increases in the length of polyproteins that contain disulfide bonds and that are stretched at a constant force with the cantilever of an atomic force microscope (AFM). The kinetics of this reaction has been measured from single-exponential fits to ensemble averages of the reduction events. However, exponential fits are notoriously ambiguous to use in cases of kinetic data showing multiple reaction pathways. Here we introduce a dwell time analysis technique, of widespread use in the single ion channel field, that we apply to the examination of the kinetics of reduction of disulfide bonds measured from single-molecule force-clamp spectroscopy traces. In this technique, exponentially distributed dwell time data is plotted as a histogram with a logarithmic time scale and a square root ordinate. The advantage of logarithmic histograms is that exponentially distributed dwell times appear as well-defined peaks in the distribution, greatly enhancing our ability to detect multiple kinetic pathways. We apply this technique to examine the distribution of dwell times of 4488 single disulfide bond reduction events measured in the presence of two very different kinds of reducing agents: tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) and the enzyme thioredoxin (TRX). A different clamping force is used for each reducing agent to obtain distributions of dwell times on a similar time scale. In the case of TCEP, the logarithmic histogram of dwell times showed a single peak, corresponding to a single reaction mechanism. By contrast, similar experiments done with TRX showed two well-separated peaks, marking two distinct modes of chemical reduction operating simultaneously. These experiments demonstrate that dwell time analysis techniques are a powerful approach to studying chemical reactions at the single-molecule level.

Thioredoxins are enzymes that catalyse disulphide bond reduction in all living organisms. Although catalysis is thought to proceed through a substitution nucleophilic bimolecular (S(N)2) reaction, the role of the enzyme in modulating this chemical reaction is unknown. Here, using single-molecule force-clamp spectroscopy, we investigate the catalytic mechanism of Escherichia coli thioredoxin (Trx). We applied mechanical force in the range of 25-600 pN to a disulphide bond substrate and monitored the reduction of these bonds by individual enzymes. We detected two alternative forms of the catalytic reaction, the first requiring a reorientation of the substrate disulphide bond, causing a shortening of the substrate polypeptide by 0.79 +/- 0.09 A (+/- s.e.m.), and the second elongating the substrate disulphide bond by 0.17 +/- 0.02 A (+/- s.e.m.). These results support the view that the Trx active site regulates the geometry of the participating sulphur atoms with sub-ångström precision to achieve efficient catalysis. Our results indicate that substrate conformational changes may be important in the regulation of Trx activity under conditions of oxidative stress and mechanical injury, such as those experienced in cardiovascular disease. Furthermore, single-molecule atomic force microscopy techniques, as shown here, can probe dynamic rearrangements within an enzyme's active site during catalysis that cannot be resolved with any other current structural biological technique.

The analysis of correlated mutations in protein sequence alignments is of considerable interest, since it may provide useful energetic and even structural information (ideally, residue contacts). However, a number of recent experimental studies support the existence of long-distance communication in proteins, a fact that may lead to correlation between distant residues. We introduce in this work a simple statistical procedure to describe the relation structure--alignments on the basis of the residue--residue distance dependence of the number of residue couples over given thresholds of a correlation measure (such as a covariance value). This procedure may lead to clear pictures of the distance distribution of correlated mutations and may provide a simple but efficient tool to explore the different structural features that are reflected in the sequence alignments.

Plants contain several genes encoding thioredoxin h. In cereals, type-h thioredoxins are abundant in developing and germinating grains, but the mechanism regulating the expression of these genes and their specific function is poorly known. The cloning of three full-length cDNAs encoding thioredoxin h, stated Trxh1, Trxh2 and Trxh3, from wheat (Triticum aestivum cv. Soissons) seeds is described here. TRXh2 and TRXh3 deduced proteins show high identity between them and with other thioredoxins h previously described from wheat, and contain exclusively the two Cys residues forming part of the active site. By contrast, TRXh1 shows a lower level of identity and contains an additional Cys residue. The three wheat thioredoxins were expressed in E. coli and their activity was demonstrated using both the DTT-dependent insulin assay and a coupled assay with recombinant NTR from wheat. Site-directed mutagenesis showed that the additional Cys residue of TRXh1 has a low effect on its activity but is essential for dimerization. Specific expression of the three thioredoxin genes was analysed by real-time RT-PCR in developing and germinating seeds and seedlings under stressed and unstressed conditions. An increase of Trxh1, Trxh2, and Trxh3 transcripts was detected at the beginning of the desiccation phase during seed development. Early after imbibition, Trxh1, but not Trxh2 or Trxh3, transcripts showed a transient increase. Treatment of wheat seedlings with salt or hydrogen peroxide caused a differential pattern of expression of the three Trxh genes between and within tissues, hence suggesting specific functions for these thioredoxins during germination and early seedling growth.

Technological applications of proteins are often hampered by their low-stability and, consequently, the development of procedures for protein stabilization is of considerable biotechnological interest. Here, we use simple electrostatics to determine positions in E. coli thioredoxin at which mutations that introduce new charged residues are expected to lead to stability enhancement. We also obtain the corresponding mutants and characterize their stability using differential scanning calorimetry. The results are interpreted in terms of the accessibility in the native structure of the mutated residues and the potential effect of the mutations on the residual structure of the denatured state.

Understanding the screening by salts of charge-charge interactions in proteins is important for at least two reasons: a), screening by intracellular salt concentration may modulate the stability and interactions of proteins in vivo; and b), the in vitro experimental estimation of the contributions from charge-charge interactions to molecular processes involving proteins is generally carried out on the basis of the salt effect on process energetics, under the assumption that these interactions are screened out by moderate salt concentrations. Here, we explore experimentally the extent to which the screening efficiency depends on the nature of the salt. To this end, we have carried out an energetic characterization of the effect of NaCl (a nondenaturing salt), guanidinium chloride (a denaturing salt), and guanidinium thiocyanate (a stronger denaturant) on the stability of the wild-type form and a T14K variant of Escherichia coli thioredoxin. Our results suggest that the efficiency of different salts to screen charge-charge interactions correlates with their denaturing strength and with the position of the constituent ions in the Hofmeister rankings. This result appears consistent with the plausible relation of the Hofmeister rankings with the extent of solute accumulation/exclusion from protein surfaces.

Thioredoxins h are ubiquitous proteins reduced by NADPH- thioredoxin reductase (NTR). They are able to reduce disulphides in target proteins. In monocots, thioredoxins h accumulate at high level in seeds and show a predominant localization in the nucleus of seed cells. These results suggest that the NTR-thioredoxin h system probably plays an important role in seed physiology. To date, the study of this system in monocots is limited by the lack of information about NTR. In the present study, we describe the cloning of a full-length cDNA encoding NTR from wheat ( Triticum aestivum ). The polypeptide deduced from this cDNA shows close similarity to NTRs from Arabidopsis, contains FAD- and NADPH-binding domains and a disulphide probably interacting with the disulphide at the active site of thioredoxin h. Wheat NTR was expressed in Escherichia coli as a His-tagged protein. The absorption spectrum of the purified recombinant protein is typical of flavoenzymes. Furthermore, it showed NADPH-dependent thioredoxin h reduction activity, thus confirming that the cDNA clone reported in the present study encodes wheat NTR. Using the His-tagged NTR and TRXhA (wheat thioredoxin h ), we successfully reconstituted the wheat NTR-thioredoxin h system in vitro, as shown by the insulin reduction assay. A polyclonal antibody was raised against wheat NTR after immunization of rabbits with the purified His-tagged protein. This antibody efficiently detected a single polypeptide of the corresponding molecular mass in seed extracts and it allowed the analysis of the pattern of accumulation of NTR in different wheat organs and developmental stages. NTR shows a wide distribution in wheat, but, surprisingly, its accumulation in seeds is low, in contrast with the level of thioredoxins h.

We have calculated the absolute heat capacities of fragments 1--73 (N fragment) and 74--108 (C fragment) from thioredoxin, their complex and the uncleaved protein, from the concentration dependence of the apparent heat capacities of the solutions determined by differential scanning calorimetry. We find that, while the absolute heat capacities of uncleaved, unfolded thioredoxin and the C fragment are in good agreement with the theoretical values expected for fully solvated chains (calculated as the sum of the contributions of the constituent amino acids), the absolute heat capacities of the N fragment and the unfolded complex are about 2 kJ x K(-1) x mol(-1) lower than the fully solvated-chain values. We attribute this discrepancy to burial of the apolar surface in the N fragment (as burial of the polar area is expected to lead to an increase in heat capacity). Illustrative calculations suggest that burial of about 1000--1600 A(2) of apolar surface takes place in the N fragment (probably accompanied by the burial of a smaller amount of polar surface). In general, this work is supportive of heat capacity measurements on protein fragments being useful as probes of surface burial in studies to characterize protein unfolded states and the high regions of protein folding landscapes.