Protein folding drives disulfide formation.

PDI catalyzes the oxidative folding of disulfide-containing proteins. However, the sequence of reactions leading to a natively folded and oxidized protein remains unknown. Here we demonstrate a technique that enables independent measurements of disulfide formation and protein folding. We find that non-native disulfides are formed early in the folding pathway and can trigger misfolding. In contrast, a PDI domain favors native disulfides by catalyzing oxidation at a late stage of folding. We propose a model for cotranslational oxidative folding wherein PDI acts as a placeholder that is relieved by the pairing of cysteines caused by substrate folding. This general mechanism can explain how PDI catalyzes oxidative folding in a variety of structurally unrelated substrates.

Protection of Catalytic Cofactors by Polypeptides as a Driver for the Emergence of Primordial Enzymes.

Enzymes catalyze the chemical reactions of life. For nearly half of known enzymes, catalysis requires the binding of small molecules known as cofactors. Polypeptide-cofactor complexes likely formed at a primordial stage and became starting points for the evolution of many efficient enzymes. Yet, evolution has no foresight so the driver for the primordial complex formation is unknown. Here, we use a resurrected ancestral TIM-barrel protein to identify one potential driver. Heme binding at a flexible region of the ancestral structure yields a peroxidation catalyst with enhanced efficiency when compared to free heme. This enhancement, however, does not arise from protein-mediated promotion of catalysis. Rather, it reflects the protection of bound heme from common degradation processes and a resulting longer lifetime and higher effective concentration for the catalyst. Protection of catalytic cofactors by polypeptides emerges as a general mechanism to enhance catalysis and may have plausibly benefited primordial polypeptide-cofactor associations.

Cell Survival Enabled by Leakage of a Labile Metabolic Intermediate.

Many metabolites are generated in one step of a biochemical pathway and consumed in a subsequent step. Such metabolic intermediates are often reactive molecules which, if allowed to freely diffuse in the intracellular milieu, could lead to undesirable side reactions and even become toxic to the cell. Therefore, metabolic intermediates are often protected as protein-bound species and directly transferred between enzyme active sites in multi-functional enzymes, multi-enzyme complexes, and metabolons. Sequestration of reactive metabolic intermediates thus contributes to metabolic efficiency. It is not known, however, whether this evolutionary adaptation can be relaxed in response to challenges to organismal survival. Here, we report evolutionary repair experiments on Escherichia coli cells in which an enzyme crucial for the biosynthesis of proline has been deleted. The deletion makes cells unable to grow in a culture medium lacking proline. Remarkably, however, cell growth is efficiently restored by many single mutations (12 at least) in the gene of glutamine synthetase. The mutations cause the leakage to the intracellular milieu of a highly reactive phosphorylated intermediate common to the biosynthetic pathways of glutamine and proline. This intermediate is generally assumed to exist only as a protein-bound species. Nevertheless, its diffusion upon mutation-induced leakage enables a new route to proline biosynthesis. Our results support that leakage of sequestered metabolic intermediates can readily occur and contribute to organismal adaptation in some scenarios. Enhanced availability of reactive molecules may enable the generation of new biochemical pathways and the potential of mutation-induced leakage in metabolic engineering is noted.

Immunoanalytical Approach for Detecting and Identifying Ancestral Peptide Biomarkers in Early Earth Analogue Environments.

Several mass spectrometry and spectroscopic techniques have been used in the search for molecular biomarkers on Mars. A major constraint is their capability to detect and identify large and complex compounds such as peptides or other biopolymers. Multiplex immunoassays can detect these compounds, but antibodies must be produced for a large number of sequence-dependent molecular targets. Ancestral Sequence Reconstruction (ASR) followed by protein "resurrection" in the lab can help to narrow the selection of targets. Herein, we propose an immunoanalytical method to identify ancient and universally conserved protein/peptide sequences as targets for identifying ancestral biomarkers in nature. We have developed, tested, and validated this approach by producing antibodies to eight previously described ancestral resurrected proteins (three ß-lactamases, three thioredoxins, one Elongation Factor Tu, and one RuBisCO, all of them theoretically dated as Precambrian), and used them as a proxy to search for any potential feature of them that could be present in current natural environments. By fluorescent sandwich microarray immunoassays (FSMI), we have detected positive immunoreactions with antibodies to the oldest ß-lactamase and thioredoxin proteins (ca. 4 Ga) in samples from a hydrothermal environment. Fine epitope mapping and inhibitory immunoassays allowed the identification of well-conserved epitope peptide sequences that resulted from ASR and were present in the sample. We corroborated these results by metagenomic sequencing and found several genes encoding analogue proteins with significant matches to the peptide epitopes identified with the antibodies. The results demonstrated that peptides inferred from ASR studies have true counterpart analogues in Nature, which validates and strengthens the well-known ASR/protein resurrection technique and our immunoanalytical approach for investigating ancient environments and metabolisms on Earth and elsewhere.

Efficient Base-Catalyzed Kemp Elimination in an Engineered Ancestral Enzyme.

The routine generation of enzymes with completely new active sites is a major unsolved problem in protein engineering. Advances in this field have thus far been modest, perhaps due, at least in part, to the widespread use of modern natural proteins as scaffolds for de novo engineering. Most modern proteins are highly evolved and specialized and, consequently, difficult to repurpose for completely new functionalities. Conceivably, resurrected ancestral proteins with the biophysical properties that promote evolvability, such as high stability and conformational diversity, could provide better scaffolds for de novo enzyme generation. Kemp elimination, a non-natural reaction that provides a simple model of proton abstraction from carbon, has been extensively used as a benchmark in de novo enzyme engineering. Here, we present an engineered ancestral ß-lactamase with a new active site that is capable of efficiently catalyzing Kemp elimination. The engineering of our Kemp eliminase involved minimalist design based on a single function-generating mutation, inclusion of an extra polypeptide segment at a position close to the de novo active site, and sharply focused, low-throughput library screening. Nevertheless, its catalytic parameters (kcat/KM~2·105 M-1 s-1, kcat~635 s-1) compare favorably with the average modern natural enzyme and match the best proton-abstraction de novo Kemp eliminases that are reported in the literature. The general implications of our results for de novo enzyme engineering are discussed.

Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases.

Ancestral sequence reconstruction and resurrection provides useful information for protein engineering, yet its alliance with directed evolution has been little explored. In this study, we have resurrected several ancestral nodes of fungal laccases dating back ∼500 to 250 million years. Unlike modern laccases, the resurrected Mesozoic laccases were readily secreted by yeast, with similar kinetic parameters, a broader stability, and distinct pH activity profiles. The resurrected Agaricomycetes laccase carried 136 ancestral mutations, a molecular testimony to its origin, and it was subjected to directed evolution in order to improve the rate of 1,3-cyclopentanedione oxidation, a ß-diketone initiator commonly used in vinyl polymerization reactions.IMPORTANCE The broad variety of biotechnological uses of fungal laccases is beyond doubt (food, textiles, pulp and paper, pharma, biofuels, cosmetics, and bioremediation), and protein engineering (in particular, directed evolution) has become the key driver for adaptation of these enzymes to harsh industrial conditions. Usually, the first requirement for directed laccase evolution is heterologous expression, which presents an important hurdle and often a time-consuming process. In this work, we resurrected a fungal Mesozoic laccase node which showed strikingly high heterologous expression and pH stability. As a proof of concept that the ancestral laccase is a suitable blueprint for engineering, we performed a quick directed evolution campaign geared to the oxidation of the ß-diketone 1,3-cyclopentanedione, a poor laccase substrate that is used in the polymerization of vinyl monomers.

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.

Engineering ancestral protein hyperstability.

Many experimental analyses and proposed scenarios support that ancient life was thermophilic. In congruence with this hypothesis, proteins encoded by reconstructed sequences corresponding to ancient phylogenetic nodes often display very high stability. Here, we show that such 'reconstructed ancestral hyperstability' can be further engineered on the basis of a straightforward approach that uses exclusively information afforded by the ancestral reconstruction process itself. Since evolution does not imply continuous progression, screening of the mutations between two evolutionarily related resurrected ancestral proteins may identify mutations that further stabilize the most stable one. To explore this approach, we have used a resurrected thioredoxin corresponding to the last common ancestor of the cyanobacterial, Deinococcus and Thermus groups (LPBCA thioredoxin), which has a denaturation temperature of ∼123°C. This high value is within the top 0.1% of the denaturation temperatures in the ProTherm database and, therefore, achieving further stabilization appears a priori as a challenging task. Nevertheless, experimental comparison with a resurrected thioredoxin corresponding to the last common ancestor of bacteria (denaturation temperature of ∼115°C) immediately identifies three mutations that increase the denaturation temperature of LPBCA thioredoxin to ∼128°C. Comparison between evolutionarily related resurrected ancestral proteins thus emerges as a simple approach to expand the capability of ancestral reconstruction to search sequence space for extreme protein properties of biotechnological interest. The fact that ancestral sequences for many phylogenetic nodes can be derived from a single alignment of modern sequences should contribute to the general applicability of this approach.

Evolution of conformational dynamics determines the conversion of a promiscuous generalist into a specialist enzyme.

ß-Lactamases are produced by many modern bacteria as a mechanism of resistance toward ß-lactam antibiotics, the most common antibiotics in use. ß-Lactamases, however, are ancient enzymes that originated billions of years ago. Recently, proteins corresponding to 2- to 3-Gy-old Precambrian nodes in the evolution of Class A ß-lactamases have been prepared and shown to be moderately efficient promiscuous catalysts, able to degrade a variety of antibiotics with catalytic efficiency levels similar to those of an average modern enzyme. Remarkably, there are few structural differences (in particular at the active-site regions) between the resurrected enzymes and a penicillin-specialist modern ß-lactamase. Here, we propose that the ancestral promiscuity originates from conformational dynamics. We investigate the differences in conformational dynamics of the ancient and extant ß-lactamases through MD simulations and quantify the contribution of each position to functionally related dynamics through Dynamic Flexibility Index. The modern TEM-1 lactamase shows a comparatively rigid active-site region, likely reflecting adaptation for efficient degradation of a specific substrate (penicillin), whereas enhanced deformability at the active-site neighborhood in the ancestral resurrected proteins likely accounts for the binding and subsequent degradation of antibiotic molecules of different size and shape. Clustering of the conformational dynamics on the basis of Principal Component Analysis is in agreement with the functional divergence, as the ancient ß-lactamases cluster together, separated from their modern descendant. Finally, our analysis leads to testable predictions, as sites of potential relevance for the evolution of dynamics are identified and mutations at those sites are expected to alter substrate-specificity.

Mutational studies on resurrected ancestral proteins reveal conservation of site-specific amino acid preferences throughout evolutionary history.

Local protein interactions ("molecular context" effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling. Here, we take advantage of the recent availability of phenotypically supported laboratory resurrections of Precambrian thioredoxins and ß-lactamases to experimentally address the change of site-specific amino acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary adjustment to a new amino acid may occur, but to a large extent this is insufficient to erase the primitive preference for amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evolutionary history despite local sequence divergence. We show such preference conservation to be readily understandable in molecular terms and we provide crystallographic evidence for an intriguing structural-switch mechanism: Energetic preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation naturally leads to one plausible evolutionary explanation for the existence of intragenic global suppressor mutations.

The consensus-based approach for gene/enzyme replacement therapies and crystallization strategies: the case of human alanine-glyoxylate aminotransferase.

Protein stability is a fundamental issue in biomedical and biotechnological applications of proteins. Among these applications, gene- and enzyme-replacement strategies are promising approaches to treat inherited diseases that may benefit from protein engineering techniques, even though these beneficial effects have been largely unexplored. In the present study we apply a sequence-alignment statistics procedure (consensus-based approach) to improve the activity and stability of the human AGT (alanine-glyoxylate aminotransferase) protein, an enzyme which causes PH1 (primary hyperoxaluria type I) upon mutation. By combining only five consensus mutations, we obtain a variant (AGT-RHEAM) with largely enhanced in vitro thermal and kinetic stability, increased activity, and with no side effects on foldability and peroxisomal targeting in mammalian cells. The structure of AGT-RHEAM reveals changes at the dimer interface and improved electrostatic interactions responsible for increased kinetic stability. Consensus-based variants maintained the overall protein fold, crystallized more easily and improved the expression as soluble proteins in two different systems [AGT and CIPK24 (CBL-interacting serine/threonine-protein kinase) SOS2 (salt-overly-sensitive 2)]. Thus the consensus-based approach also emerges as a simple and generic strategy to increase the crystallization success for hard-to-get protein targets as well as to enhance protein stability and function for biomedical applications.

Phenotypic comparisons of consensus variants versus laboratory resurrections of Precambrian proteins.

Consensus-sequence engineering has generated protein variants with enhanced stability, and sometimes, with modulated biological function. Consensus mutations are often interpreted as the introduction of ancestral amino acid residues. However, the precise relationship between consensus engineering and ancestral protein resurrection is not fully understood. Here, we report the properties of proteins encoded by consensus sequences derived from a multiple sequence alignment of extant, class A ß-lactamases, as compared with the properties of ancient Precambrian ß-lactamases resurrected in the laboratory. These comparisons considered primary sequence, secondary, and tertiary structure, as well as stability and catalysis against different antibiotics. Out of the three consensus variants generated, one could not be expressed and purified (likely due to misfolding and/or low stability) and only one displayed substantial stability having substrate promiscuity, although to a lower extent than ancient ß-lactamases. These results: (i) highlight the phenotypic differences between consensus variants and laboratory resurrections of ancestral proteins; (ii) question interpretations of consensus proteins as phenotypic proxies of ancestral proteins; and (iii) support the notion that ancient proteins provide a robust approach toward the preparation of protein variants having large numbers of mutational changes while possessing unique biomolecular properties.

Different contribution of conserved amino acids to the global properties of triosephosphate isomerases.

It is generally assumed that the amino acids that exist in all homologous enzymes correspond to residues that participate in catalysis, or that are essential for folding and stability. Although this holds for catalytic residues, the function of conserved noncatalytic residues is not clear. It is not known if such residues are of equal importance and have the same role in different homologous enzymes. In humans, the E104D mutation in triosephosphate isomerase (TIM) is the most frequent mutation in the autosomal diseases named "TPI deficiencies." We explored if the E104D mutation has the same impact in TIMs from four different organisms (Homo sapiens, Giardia lamblia, Trypanosoma cruzi, and T. brucei). The catalytic properties were not significantly affected by the mutation, but it affected the rate and extent of formation of active dimers from unfolded monomers differently. Scanning calorimetry experiments indicated that the mutation was in all cases destabilizing, but the mutation effect on rates of irreversible denaturation and transition-state energetics were drastically dependent on the TIM background. For instance, the E104D mutation produce changes in activation energy ranging from 430 kJ mol(-1) in HsTIM to -78 kJ mol(-1) in TcTIM. Thus, in TIM the role of a conserved noncatalytic residue is drastically dependent on its molecular background. Accordingly, it would seem that because each protein has a particular sequence, and a distinctive set of amino acid interactions, it should be regarded as a unique entity that has evolved for function and stability in the organisms to which it belongs.

Human cystathionine ß-synthase (CBS) contains two classes of binding sites for S-adenosylmethionine (SAM): complex regulation of CBS activity and stability by SAM.

CBS (cystathionine ß-synthase) is a multidomain tetrameric enzyme essential in the regulation of homocysteine metabolism, whose activity is enhanced by the allosteric regulator SAM (S-adenosylmethionine). Missense mutations in CBS are the major cause of inherited HCU (homocystinuria). In the present study we apply a novel approach based on a combination of calorimetric methods, functional assays and kinetic modelling to provide structural and energetic insight into the effects of SAM on the stability and activity of WT (wild-type) CBS and seven HCU-causing mutants. We found two sets of SAM-binding sites in the C-terminal regulatory domain with different structural and energetic features: a high affinity set of two sites, probably involved in kinetic stabilization of the regulatory domain, and a low affinity set of four sites, which are involved in the enzyme activation. We show that the regulatory domain displays a low kinetic stability in WT CBS, which is further decreased in many HCU-causing mutants. We propose that the SAM-induced stabilization may play a key role in modulating steady-state levels of WT and mutant CBS in vivo. Our strategy may be valuable for understanding ligand effects on proteins with a complex architecture and their role in human genetic diseases and for the development of novel pharmacological strategies.

Hyperstability and substrate promiscuity in laboratory resurrections of Precambrian ß-lactamases.

We report a sequence reconstruction analysis targeting several Precambrian nodes in the evolution of class-A ß-lactamases and the preparation and experimental characterization of their encoded proteins. Despite extensive sequence differences with the modern enzymes (~100 amino acid differences), the proteins resurrected in the laboratory properly fold into the canonical lactamase structure. The encoded proteins from 2-3 billion years (Gyr)-old ß-lactamase sequences undergo cooperative two-state thermal denaturation and display very large denaturation temperature enhancements (~35 °C) relative to modern ß-lactamases. They degrade different antibiotics in vitro with catalytic efficiencies comparable to that of an average modern enzyme. This enhanced substrate promiscuity is not accompanied by significant changes in the active-site region as seen in static X-ray structures, suggesting a plausible role for dynamics in the evolution of function in these proteins. Laboratory resurrections of 2-3 Gyr-old ß-lactamases also endowed modern microorganisms with significant levels of resistance toward a variety of antibiotics, opening up the possibility of performing laboratory replays of the molecular tape of lactamase evolution. Overall, these results support the notions that Precambrian life was thermophilic and that proteins can evolve from substrate-promiscuous generalists into specialists during the course of natural evolution. They also highlight the biotechnological potential of laboratory resurrection of Precambrian proteins, as both high stability and enhanced promiscuity (likely contributors to high evolvability) are advantageous features in protein scaffolds for molecular design and laboratory evolution.

Probing the mutational interplay between primary and promiscuous protein functions: a computational-experimental approach.

Protein promiscuity is of considerable interest due its role in adaptive metabolic plasticity, its fundamental connection with molecular evolution and also because of its biotechnological applications. Current views on the relation between primary and promiscuous protein activities stem largely from laboratory evolution experiments aimed at increasing promiscuous activity levels. Here, on the other hand, we attempt to assess the main features of the simultaneous modulation of the primary and promiscuous functions during the course of natural evolution. The computational/experimental approach we propose for this task involves the following steps: a function-targeted, statistical coupling analysis of evolutionary data is used to determine a set of positions likely linked to the recruitment of a promiscuous activity for a new function; a combinatorial library of mutations on this set of positions is prepared and screened for both, the primary and the promiscuous activities; a partial-least-squares reconstruction of the full combinatorial space is carried out; finally, an approximation to the Pareto set of variants with optimal primary/promiscuous activities is derived. Application of the approach to the emergence of folding catalysis in thioredoxin scaffolds reveals an unanticipated scenario: diverse patterns of primary/promiscuous activity modulation are possible, including a moderate (but likely significant in a biological context) simultaneous enhancement of both activities. We show that this scenario can be most simply explained on the basis of the conformational diversity hypothesis, although alternative interpretations cannot be ruled out. Overall, the results reported may help clarify the mechanisms of the evolution of new functions. From a different viewpoint, the partial-least-squares-reconstruction/Pareto-set-prediction approach we have introduced provides the computational basis for an efficient directed-evolution protocol aimed at the simultaneous enhancement of several protein features and should therefore open new possibilities in the engineering of multi-functional enzymes.

Probing the chemistry of thioredoxin catalysis with force.

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.

On promiscuity, changing environments and the possibility of replaying the molecular tape of life.

The consequences for organism fitness of mutations in a given protein are often thought to be determined to a significant extent by epistasis, that is, by the fact that the effect of a mutation may be strongly dependent on the previous mutational background. Actually, a given mutation could be deleterious or beneficial depending on the background, a situation known as 'sign epistasis'. Under pervasive sign epistasis, many mutational trajectories towards a 'fitter protein' will show a 'dip' in fitness and, it has been previously suggested, only a few trajectories will be available to Darwinian selection. In this issue of the Biochemical Journal, Zhang et al. explore how this simple picture needs to be modified when two rather general and important features are taken into account, namely that many proteins are promiscuous and that living organisms must survive and thrive in environments that change continuously. The multidimensional nature of epistasis for a protein involved in several tasks, together with the fact that different tasks may become critical for organism survival as environmental conditions change, is shown by Zhang et al. to contribute to eliminating fitness dead-ends in protein sequence space. Consequently, many alternative mutational trajectories should allow protein optimization for enhanced organism fitness under changing environmental conditions.