Stable monomers in the ancestral sequence reconstruction of the last opisthokont common ancestor of dimeric triosephosphate isomerase.

Function and structure are strongly coupled in obligated oligomers such as Triosephosphate isomerase (TIM). In animals and fungi, TIM monomers are inactive and unstable. Previously, we used ancestral sequence reconstruction to study TIM evolution and found that before these lineages diverged, the last opisthokonta common ancestor of TIM (LOCATIM) was an obligated oligomer that resembles those of extant TIMs. Notably, calorimetric evidence indicated that ancestral TIM monomers are more structured than extant ones. To further increase confidence about the function, structure, and stability of the LOCATIM, in this work, we applied two different inference methodologies and the worst plausible case scenario for both of them, to infer four sequences of this ancestor and test the robustness of their physicochemical properties. The extensive biophysical characterization of the four reconstructed sequences of LOCATIM showed very similar hydrodynamic and spectroscopic properties, as well as ligand-binding energetics and catalytic parameters. Their 3D structures were also conserved. Although differences were observed in melting temperature, all LOCATIMs showed reversible urea-induced unfolding transitions, and for those that reached equilibrium, high conformational stability was estimated (ΔGTot = 40.6-46.2 kcal/mol). The stability of the inactive monomeric intermediates was also high (ΔGunf = 12.6-18.4 kcal/mol), resembling some protozoan TIMs rather than the unstable monomer observed in extant opisthokonts. A comparative analysis of the 3D structure of ancestral and extant TIMs shows a correlation between the higher stability of the ancestral monomers with the presence of several hydrogen bonds located in the "bottom" part of the barrel.

Thermodynamic and kinetic analysis of the LAO binding protein and its isolated domains reveal non-additivity in stability, folding and function.

Substrate-binding proteins (SBPs) are used by organisms from the three domains of life for transport and signalling. SBPs are composed of two domains that collectively trap ligands with high affinity and selectivity. To explore the role of the domains and the integrity of the hinge region between them in the function and conformation of SBPs, here, we describe the ligand binding, conformational stability and folding kinetics of the Lysine Arginine Ornithine (LAO) binding protein from Salmonella thiphimurium and constructs corresponding to its two independent domains. LAO is a class II SBP formed by a continuous and a discontinuous domain. Contrary to the expected behaviour based on their connectivity, the discontinuous domain shows a stable native-like structure that binds l-arginine with moderate affinity, whereas the continuous domain is barely stable and shows no detectable ligand binding. Regarding folding kinetics, studies of the entire protein revealed the presence of at least two intermediates. While the unfolding and refolding of the continuous domain exhibited only a single intermediate and simpler and faster kinetics than LAO, the folding mechanism of the discontinuous domain was complex and involved multiple intermediates. These findings suggest that in the complete protein the continuous domain nucleates folding and that its presence funnels the folding of the discontinuous domain avoiding nonproductive interactions. The strong dependence of the function, stability and folding pathway of the lobes on their covalent association is most likely the result of the coevolution of both domains as a single unit.

Swim Bladder of Farmed Totoaba macdonaldi: A Source of Value-Added Collagen.

Finding strategies to use the swim bladder of farmed totoaba (Totoaba macdonaldi) is of the utmost need to reduce waste. Fish swim bladders are rich in collagen; hence, extracting collagen is a promising alternative with benefits for aquaculture of totoaba and the environment. The elemental biochemical composition of totoaba swim bladders, including their proximate and amino acid compositions, was determined. Pepsin-soluble collagen (PSC) was used to extract collagen from swim bladders, and its characteristics were analyzed. Alcalase and papain were used for the preparation of collagen hydrolysates. Swim bladders contained 95% protein, 2.4% fat, and 0.8% ash (on a dry basis). The essential amino acid content was low, but the functional amino acid content was high. The PSC yield was high, at 68% (dry weight). The amino acid composition profile, electrophoretic pattern, and structural integrity analyses of the isolated collagen suggested it is a typical type-I collagen with high purity. The denaturalization temperature was 32.5 °C, probably attributable to the imino acid content (205 residues/1000 residues). Papain-hydrolysates (≤3 kDa) of this collagen exhibited higher radical scavenging activity than Alcalase-hydrolysates. The swim bladder from the farmed totoaba could be an ideal source to produce high-quality type I collagen and may be considered an alternative to conventional collagen sources or bioactive peptides.

Engineering Concanavalin B to Release Bioactive Peptides against Metabolic Syndrome.

Metabolic syndrome is a severe public health issue characterized by multiple metabolic disturbances. Current treatments prescribe a particular drug for each of them, producing multiple side effects. As a first step towards a more integral approach, we applied our recently described methodology to design single proteins, based in the Concanavalin B scaffold (1CNV), that contain several bioactive peptides (BPs), including antioxidant and lipid-lowering activities as well as inhibitors of dipeptidyl peptidase IV (DPPIV) and the angiotensin converting enzyme. Modified Concanavalin (CNV44), the designed protein that showed the best in silico properties, was expressed in high yields in E. coli and purified to homogeneity. After in vitro digestion with gastrointestinal enzymes, all the biological activities tested where higher in CNV44 when compared to the non-modified protein 1CNV, or to other previous reports. The results presented here represent the first in vitro evidence of a modified protein with the potential to treat metabolic syndrome and open the venue for the design of proteins to treat other non-communicable diseases.

The Stability Landscape of de novo TIM Barrels Explored by a Modular Design Approach.

The ability to design stable proteins with custom-made functions is a major goal in biochemistry with practical relevance for our environment and society. Understanding and manipulating protein stability provide crucial information on the molecular determinants that modulate structure and stability, and expand the applications of de novo proteins. Since the (ß/⍺)8-barrel or TIM-barrel fold is one of the most common functional scaffolds, in this work we designed a collection of stable de novo TIM barrels (DeNovoTIMs), using a computational fixed-backbone and modular approach based on improved hydrophobic packing of sTIM11, the first validated de novo TIM barrel, and subjected them to a thorough folding analysis. DeNovoTIMs navigate a region of the stability landscape previously uncharted by natural TIM barrels, with variations spanning 60 degrees in melting temperature and 22 kcal per mol in conformational stability throughout the designs. Significant non-additive or epistatic effects were observed when stabilizing mutations from different regions of the barrel were combined. The molecular basis of epistasis in DeNovoTIMs appears to be related to the extension of the hydrophobic cores. This study is an important step towards the fine-tuned modulation of protein stability by design.

The Folding Pathway of 6aJL2.

One-third of the reported cases of light chain amyloidosis are related to the germ line λ6 family; remarkably, healthy individuals express this type of protein in just 2% of the peripheral blood and bone marrow B-cells. The appearance of the disease has been related to the inherent properties of this protein family. A recombinant representative model for λ6 proteins called 6aJL2 containing the amino acid sequence encoded by the 6a and JL2 germ line genes was previously designed and synthesized to study the properties of this family. Previous work on 6aJL2 suggested a simple two-state folding model at 25 °C; no intermediate could be identified either by kinetics or by fluorescence and circular dichroism equilibrium studies, although the presence of an intermediate that is populated at ∼2.4 M urea was suggested by size exclusion chromatography. In this study we employed classic equilibrium and kinetic experiments and analysis to elucidate the detailed folding mechanism of this protein. We identify species that are kinetically accessible and/or are populated at equilibrium. We describe the presence of intermediate and native-like species and propose a five-species folding mechanism at 25 °C at short incubation times, similar to and consistent with those observed in other proteins of this fold. The formation of intermediates in the mechanism of 6aJL2 is faster than that proposed for a Vκ light chain, which could be an important distinction in the amyloidogenic potential of both germ lines.

The insertion of bioactive peptides at the C-terminal end of an 11S globulin changes the structural stability and improves the antihypertensive activity

BACKGROUND: The 11S globulin from amaranth is the most abundant storage protein in mature seeds and is well recognized for its nutritional value. We used this globulin to engineer a new protein by adding a four valinetyrosine antihypertensive peptide at its C-terminal end to improve its functionality. The new protein was named AMR5 and expressed in the Escherichia coli BL21-CodonPlus(DE3)-RIL strain using a custom medium (F8PW) designed for this work. RESULTS: The alternative medium allowed for the production of 652 mg/L expressed protein at the flask level, mostly in an insoluble form, and this protein was subjected to in vitro refolding. The spectrometric analysis suggests that the protein adopts a ß/α structure with a small increment of α-helix conformation relative to the native amaranth 11S globulin. Thermal and urea denaturation experiments determined apparent Tm and C1/2 values of 50.4°C and 3.04 M, respectively, thus indicating that the antihypertensive peptide insertion destabilized the modified protein relative to the native one. AMR5 hydrolyzed by trypsin and chymotrypsin showed 14- and 1.3-fold stronger inhibitory activity against angiotensin I-converting enzyme (IC50 of 0.034 mg/mL) than the unmodified protein and the previously reported amaranth acidic subunit modified with antihypertensive peptides, respectively. CONCLUSION: The inserted peptide decreases the structural stability of amaranth 11S globulin and improves its antihypertensive activity.

Structural basis for the modulation of plant cytosolic triosephosphate isomerase activity by mimicry of redox-based modifications.

Reactive oxidative species (ROS) and S-glutathionylation modulate the activity of plant cytosolic triosephosphate isomerases (cTPI). Arabidopsis thaliana cTPI (AtcTPI) is subject of redox regulation at two reactive cysteines that function as thiol switches. Here we investigate the role of these residues, AtcTPI-Cys13 and At-Cys218, by substituting them with aspartic acid that mimics the irreversible oxidation of cysteine to sulfinic acid and with amino acids that mimic thiol conjugation. Crystallographic studies show that mimicking AtcTPI-Cys13 oxidation promotes the formation of inactive monomers by reposition residue Phe75 of the neighboring subunit, into a conformation that destabilizes the dimer interface. Mutations in residue AtcTPI-Cys218 to Asp, Lys, or Tyr generate TPI variants with a decreased enzymatic activity by creating structural modifications in two loops (loop 7 and loop 6) whose integrity is necessary to assemble the active site. In contrast with mutations in residue AtcTPI-Cys13, mutations in AtcTPI-Cys218 do not alter the dimeric nature of AtcTPI. Therefore, modifications of residues AtcTPI-Cys13 and AtcTPI-Cys218 modulate AtcTPI activity by inducing the formation of inactive monomers and by altering the active site of the dimeric enzyme, respectively. The identity of residue AtcTPI-Cys218 is conserved in the majority of plant cytosolic TPIs, this conservation and its solvent-exposed localization make it the most probable target for TPI regulation upon oxidative damage by reactive oxygen species. Our data reveal the structural mechanisms by which S-glutathionylation protects AtcTPI from irreversible chemical modifications and re-routes carbon metabolism to the pentose phosphate pathway to decrease oxidative stress.

Insertion of antihypertensive peptides in acidic subunit from amaranth 11S induces contrasting effects in stability.

The insertion of peptides is a biotechnology tool widely used to improve the nutraceutical properties of proteins. Because the effect of these insertions in protein stability and function is difficult to predict, it should be determined experimentally. In this study, we created two variants of amarantin acidic subunit and analyzed them along with other four proteins reported previously. We measured their response against two destabilizing agents: temperature and urea. The six proteins presented the insertion of antihypertensive peptides (VYVYVYVY or RIPP) in the variable regions of the protein. We observed that their effect strongly depended on the site of the insertion. The insertion in the variable region I stabilized the protein both thermally and chemically, but it affected the inhibitory activity of the angiotensin-converting enzyme in vitro. In contrast, insertions in other three regions were severely destabilizing, producing molten globules. Our findings reveal that the insertion of bioactive peptides in variable regions of a protein can increase or decrease the protein's thermal and chemical stability and that these conformational changes may also alter its final activity.

Structure and conformational stability of the triosephosphate isomerase from Zea mays. Comparison with the chemical unfolding pathways of other eukaryotic TIMs.

We studied the structure, function and thermodynamic properties for the unfolding of the Triosephosphate isomerase (TIM) from Zea mays (ZmTIM). ZmTIM shows a catalytic efficiency close to the diffusion limit. Native ZmTIM is a dimer that dissociates upon dilution into inactive and unfolded monomers. Its thermal unfolding is irreversible with a Tm of 61.6 ±â€¯1.4 °C and an activation energy of 383.4 ±â€¯11.5 kJ mol-1. The urea-induced unfolding of ZmTIM is reversible. Transitions followed by catalytic activity and spectroscopic properties are monophasic and superimposable, indicating that ZmTIM unfolds/refolds in a two-state behavior with an unfolding ΔG°(H20) = 99.8 ±â€¯5.3 kJ mol-1. This contrasts with most other studied TIMs, where folding intermediates are common. The three-dimensional structure of ZmTIM was solved at 1.8 Å. A structural comparison with other eukaryotic TIMs shows a similar number of intramolecular and intermolecular interactions. Interestingly the number of interfacial water molecules found in ZmTIM is lower than those observed in most TIMs that show folding intermediates. Although with the available data, there is no clear correlation between structural properties and the number of equilibrium intermediates in the unfolding of TIM, the identification of such structural properties should increase our understanding of folding mechanisms.

Localized conformational changes trigger the pH-induced fibrillogenesis of an amyloidogenic λ light chain protein.

Solvent conditions modulate the expression of the amyloidogenic potential of proteins. In this work the effect of pH on the fibrillogenic behavior and the conformational properties of 6aJL2, a model protein of the highly amyloidogenic variable light chain λ6a gene segment, was examined. Ordered aggregates showing the ultrastructural and spectroscopic properties observed in amyloid fibrils were formed in the 2.0-8.0 pH range. At pH <3.0 a drastic decrease in lag time and an increase in fibril formation rate were found. In the 4.0-8.0 pH range there was no spectroscopic evidence for significant conformational changes in the native state. Likewise, heat capacity measurements showed no evidence for residual structure in the unfolded state. However, at pH <3.0 stability is severely decreased and the protein suffers conformational changes as detected by circular dichroism, tryptophan and ANS fluorescence, as well as by NMR spectroscopy. Molecular dynamics simulations indicate that acid-induced conformational changes involve the exposure of the loop connecting strands E and F. These results are compatible with pH-induced changes in the NMR spectra. Overall, the results indicate that the mechanism involved in the acid-induced increase in the fibrillogenic potential of 6aJL2 is profoundly different to that observed in κ light chains, and is promoted by localized conformational changes in a region of the protein that was previously not known to be involved in acid-induced light chain fibril formation. The identification of this region opens the potential for the design of specific inhibitors.

Massively parallel de novo protein design for targeted therapeutics.

De novo protein design holds promise for creating small stable proteins with shapes customized to bind therapeutic targets. We describe a massively parallel approach for designing, manufacturing and screening mini-protein binders, integrating large-scale computational design, oligonucleotide synthesis, yeast display screening and next-generation sequencing. We designed and tested 22,660 mini-proteins of 37-43 residues that target influenza haemagglutinin and botulinum neurotoxin B, along with 6,286 control sequences to probe contributions to folding and binding, and identified 2,618 high-affinity binders. Comparison of the binding and non-binding design sets, which are two orders of magnitude larger than any previously investigated, enabled the evaluation and improvement of the computational model. Biophysical characterization of a subset of the binder designs showed that they are extremely stable and, unlike antibodies, do not lose activity after exposure to high temperatures. The designs elicit little or no immune response and provide potent prophylactic and therapeutic protection against influenza, even after extensive repeated dosing.

Stability and aggregation propensity do not fully account for the association of various germline variable domain gene segments with light chain amyloidosis.

Variable domain (VL) gene segments exhibit variable tendencies to be associated with light chain amyloidosis (AL). While few of them are very frequent in AL and give rise to most of the amyloidogenic light chains compiled at the sequence databases, other are rarely found among the AL cases. To analyze to which extent these tendencies depend on folding stability and aggregation propensity of the germline VL protein, we characterized VL proteins encoded by four AL-associated germline gene segments and one not associated to AL. We found that the AL-associated germline rVL proteins differ widely in conformational stability and propensity to in vitro amyloid aggregation. While in vitro the amyloid formation kinetics of these proteins correlate well with their folding stabilities, the folding stability does not clearly correlate with their germline's frequencies in AL. We conclude that the association of the VL genes segments to amyloidosis is not determined solely by the folding stability and aggregation propensity of the germline VL protein. Other factors, such as the frequencies of destabilizing mutations and susceptibility to proteolysis, must play a role in determining the light chain amyloidogenicity.

On the molecular basis of the high affinity binding of basic amino acids to LAOBP, a periplasmic binding protein from Salmonella typhimurium.

The rational designing of binding abilities in proteins requires an understanding of the relationship between structure and thermodynamics. However, our knowledge of the molecular origin of high-affinity binding of ligands to proteins is still limited; such is the case for l-lysine-l-arginine-l-ornithine periplasmic binding protein (LAOBP), a periplasmic binding protein from Salmonella typhimurium that binds to l-arginine, l-lysine, and l-ornithine with nanomolar affinity and to l-histidine with micromolar affinity. Structural studies indicate that ligand binding induces a large conformational change in LAOBP. In this work, we studied the thermodynamics of l-histidine and l-arginine binding to LAOBP by isothermal titration calorimetry. For both ligands, the affinity is enthalpically driven, with a binding ΔCp of ~-300 cal mol(-1) K(-1) , most of which arises from the burial of protein nonpolar surfaces that accompanies the conformational change. Osmotic stress measurements revealed that several water molecules become sequestered upon complex formation. In addition, LAOBP prefers positively charged ligands in their side chain. An energetic analysis shows that the protein acquires a thermodynamically equivalent state with both ligands. The 1000-fold higher affinity of LAOBP for l-arginine as compared with l-histidine is mainly of enthalpic origin and can be ascribed to the formation of an extra pair of hydrogen bonds. Periplasmic binding proteins have evolved diverse energetic strategies for ligand recognition. STM4351, another arginine binding protein from Salmonella, shows an entropy-driven micromolar affinity toward l-arginine. In contrast, our data show that LAOBP achieves nanomolar affinity for the same ligand through enthalpy optimization.

The N-terminal strand modulates immunoglobulin light chain fibrillogenesis.

It has been suggested that the N-terminal strand of the light chain variable domain (V(L)) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stability and kinetic of fibrillogenesis of the V(L) protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein.

Mutational and genetic determinants of λ6 light chain amyloidogenesis.

Approximately 25% of the λ6 light chains have glycine rather than arginine at position 25, which is an allelic variant of the IGLV6-57 (6a) locus. The Gly25 variant has been shown to decrease the folding stability of the germline λ6 V(L) protein 6aJL2 by 1.7 kcal·mol(-1). In this work, we compared the thermodynamic and fibrillogenic properties of the amyloidosis (AL) derived recombinant (r) V(L) protein AR, which contains the allelic variant Gly25, with those of germline rV(L) 6aJL2-R25G and the λ6 disease-associated V(L) proteins Wil (AL) and Jto (myeloma). Our experiments show that of the four proteins AR is the least stable; forms amyloid fibrils at physiological temperature, pH and ionic strength; has the shortest lag time; and elongates homologous seeds most efficiently. We conclude that the Gly25 allelic variant, together with the somatic mutations, contributes importantly to the extremely low stability and high amyloidogenicity of the AL-derived protein AR.

Thermodynamic and kinetic characterization of a germ line human lambda6 light-chain protein: the relation between unfolding and fibrillogenesis.

Proteins encoded by the gene segment 6a of the lambda variable light-chain repertoire are strongly associated with amyloid deposition. 6aJL2 is a model protein constructed with the predicted sequences encoded by the 6a and JL2 germ line genes. In this work, we characterized the urea- and temperature-induced unfolding of 6aJL2. In the short time scale, spectroscopic, hydrodynamic and calorimetric experiments were compatible with a two-state transition. Furthermore, DeltaG, m and the midpoint urea concentration obtained from equilibrium experiments were compatible with those obtained from kinetic experiments. Since fibril formation is a slow process, samples were also incubated for longer times. After incubation for several hours at 37 degrees C, spectroscopic, hydrodynamic and calorimetric experiments revealed the presence of a partially unfolded off-pathway intermediate around the midpoint urea concentration (1.5-3.0 M urea). In vitro fibrillogenesis assays show that the maximum growth rate for fibril formation and the minimum lag time were obtained at urea concentrations where the partially unfolded state was populated (2.5 M urea at 37 degrees C). This indicates that this partially unfolded state is critical for in vitro fibril formation. Concentration-dependent kinetics and hydrodynamic properties of the intermediate were consistent with a soluble oligomeric state. The intermediate is formed around the midpoint urea concentration, where the native and unfolded states are equally populated and their rate of interconversion is the slowest. This situation may promote the slow accumulation of an intermediate state that is prone to aggregate.

Thermal unfolding of triosephosphate isomerase from Entamoeba histolytica: dimer dissociation leads to extensive unfolding.

In mesophiles, triosephosphate isomerase (TIM) is an obligated homodimer. We have previously shown that monomeric folding intermediates are common in the chemical unfolding of TIM, where dissociation provides 75% of the overall conformational stability of the dimer. However, analysis of the crystallographic structure shows that, during unfolding, intermonomeric contacts contribute to only 5% of the overall increase in accessible surface area. In this work several methodologies were used to characterize the thermal dissociation and unfolding of the TIM from Entamoeba histolytica (EhTIM) and a monomeric variant obtained by chemical derivatization (mEhTIM). During EhTIM unfolding, sequential transitions corresponding to dimer dissociation into a compact monomeric intermediate followed by unfolding and further aggregation of the intermediate occurred. In the case of mEhTIM, a single transition, analogous to the second transition of EhTIM, was observed. Calorimetric, spectroscopic, hydrodynamic, and functional evidence shows that dimer dissociation is not restricted to localized interface reorganization. Dissociation represents 55% (DeltaH(Diss) = 146.8 kcal mol(-1)) of the total enthalpy change (DeltaH(Tot) = 266 kcal mol(-1)), indicating that this process is linked to substantial unfolding. We propose that, rather than a rigid body process, subunit assembly is best represented by a fly-casting mechanism. In TIM, catalysis is restricted to the dimer; therefore, the interface can be viewed as the final nucleation motif that directs assembly, folding, and function.

Hydrophobic repacking of the dimer interface of triosephosphate isomerase by in silico design and directed evolution.

Triosephosphate isomerase from Saccharomyces cerevisiae (wt-TIM) is an obligated homodimer. The interface of wt-TIM is formed by 34 residues. In the native dimer, each monomer buries nearly 2600 A(2) of accessible surface area (ASA), and 58.4% of the interface ASA is hydrophobic. We determined the thermodynamic and functional consequences of increasing the hydrophobic character of the wt-TIM interface. Mutations were restricted to a cluster of five nonconserved residues located far from the active site. Two different approaches, in silico design and directed evolution, were employed. In both methodologies, the obtained proteins were soluble, dimeric, and compact. In silico-designed proteins are very stable dimers that bind substrate with a wild-type-like K(m); albeit, they exhibited a very low k cat. Proteins obtained from directed evolution experiments show wild-type-like catalytic activity, while their stability is decreased. Hydrophobic replacements at the interface produced a remarkable shift in the dissociation step. For wt-TIM and for TIMs obtained by directed evolution, dissociation was observed in the first transition, with C(1/2) values ranging from 0.58 to 0.024 M GdnHCl, whereas for TIMs generated by in silico design, dissociation occurred in the last transition, with C(1/2) values ranging form 3.01 to 3.65 M GdnHCl. For the latter mutants, the stabilization of the interface changed the equilibrium transitions to a novel four-state process with two dimeric intermediates. The change in the intermediate nature suggests that the relative stabilities of different folding units are similar so that subtle alterations in their stability produce a total transformation of the folding pathway.