The role of Zn ions in the interaction between SARS-CoV-2 orf7a protein and BST2/tetherin.

In this paper, we provide evidence that Zn 2 + ions play a role in the SARS-CoV-2 virus strategy to escape the immune response mediated by the BST2-tetherin host protein. This conclusion is based on sequence analysis and molecular dynamics simulations as well as X-ray absorption experiments [1].

Metal Ion Binding in Wild-Type and Mutated Frataxin: A Stability Study.

This work studies the stability of wild-type frataxin and some of its variants found in cancer tissues upon Co2+ binding. Although the physiologically involved metal ion in the frataxin enzymatic activity is Fe2+, as it is customarily done, Co2+ is most often used in experiments because Fe2+ is extremely unstable owing to the fast oxidation reaction Fe2+ → Fe3+. Protein stability is monitored following the conformational changes induced by Co2+ binding as measured by circular dichroism, fluorescence spectroscopy, and melting temperature measurements. The stability ranking among the wild-type frataxin and its variants obtained in this way is confirmed by a detailed comparative analysis of the XAS spectra of the metal-protein complex at the Co K-edge. In particular, a fit to the EXAFS region of the spectrum allows positively identifying the frataxin acidic ridge as the most likely location of the metal-binding sites. Furthermore, we can explain the surprising feature emerging from a detailed analysis of the XANES region of the spectrum, showing that the longer 81-210 frataxin fragment has a smaller propensity for Co2+ binding than the shorter 90-210 one. This fact is explained by the peculiar role of the N-terminal disordered tail in modulating the protein ability to interact with the metal.

The effect of ß-sheet breaker peptides on metal associated Amyloid-ß peptide aggregation process.

Far-UV Circular Dichroism experiments and Atomic Force Microscopy tomography are employed to assess the impact of ß-sheet breakers on the Aß1-40 peptide aggregation process in the presence of Cu2+ or Zn2+ transition metals. In this work we focus on two specific 5-amino acids long ß-sheet breakers, namely the LPFFD Soto peptide, already known in the literature, and the LPFFN peptide recently designed and studied by our team. We provide evidence that both ß-sheet breakers are effective in reducing the Aß1-40 aggregation propensity, even in the presence of metal ions.

A semi-synthetic repertoire of intrinsically stable antibody fragments derived from a single-framework scaffold.

We report the design, construction and use of an antibody bacteriophage display library built on the scaffold of a single-chain variable fragment (scFv) previously proven to be functionally expressed in the reducing environment of both bacterial and plant cytoplasm and endowed with intrinsic high thermodynamic stability. Four amino acid residues of the third hypervariable loop (CDR3) of both VH and VL were combinatorially mutated, generating a repertoire of approximately 5x10(7) independent scFvs, cloned in a phagemid vector. The ability of the antibody phage library to yield specific binders was tested by biopanning against several antigens. Successful selection of fully active scFvs was obtained, confirming the notion that combinatorial mutagenesis of few amino acid residues centrally located in the antigen-binding site is sufficient to provide binding specificities against virtually any target. High yields of both soluble and phage antibodies were obtained in Escherichia coli. Maintenance of the cognate scFv antibody stability in the newly selected scFv fragments was demonstrated by guanidinium chloride denaturation/renaturation studies and by soluble antibody expression in the bacterial cytoplasm. The antibody library described here allows the isolation of new stable binding specificities, potentially exploitable as immunochemical reagents for intracellular applications.

Contribution of an aspartate residue, D114, in the active site of clostridial glutamate dehydrogenase to the enzyme's unusual pH dependence.

Glutamate dehydrogenase from Clostridium symbiosum displays unusual kinetic behaviour at high pH when compared with other members of this enzyme family. Structural and sequence comparisons with GDHs from other organisms have indicated that the Asp residue at position 114 in the clostridial enzyme may account for these differences. By replacing this residue by Asn, a mutant protein has been created with altered functional properties at high pH. This mutant protein can be efficiently overexpressed in Escherichia coli, and several criteria, including mobility in non-denaturing electrophoresis, circular dichroism (CD) spectra and initial crystallisation studies, suggest a folding and an assembly comparable to those of the wild-type protein. The D114N mutant enzyme shows a higher optimum pH for activity than the wild-type enzyme, and both CD data and activity measurements show that the distinctive time-dependent reversible conformational inactivation seen at high pH in the wild-type enzyme is abolished in the mutant.

The unusual dodecameric ferritin from Listeria innocua dissociates below pH 2.0.

The stability of the dodecameric Listeria innocua ferritin at low pH values has been investigated by spectroscopic methods and size-exclusion chromatography. The dodecamer is extremely stable in comparison to the classic ferritin tetracosamer and preserves its quaternary assembly at pH 2.0, despite an altered tertiary structure. Below pH 2.0, dissociation into dimers occurs and is paralleled by the complete loss of tertiary structure and a significant decrease in secondary structure elements. Dissociation of dimers into monomers occurs only at pH 1.0. Addition of NaCl to the protein at pH 2.0 induces structural changes similar to those observed upon increasing the proton concentration, although dissociation proceeds only to the dimer stage. Addition of sulfate at pH values >/= 1.5 prevents the dissociation of the dodecamer. The role played by hydrophilic and hydrophobic interactions in determining the resistance to dissociation of L. innocua ferritin at low pH is discussed in the light of its three-dimensional structure.

Protein stability in extremophilic archaea.

Extremophilic microorganisms have adapted their molecular machinery to grow and thrive under the most adverse environmental conditions. These microorganisms have found their natural habitat at the boiling and freezing point of water, in high salt concentration and at extreme pH values. The extremophilic proteins, selected by Nature to withstand this evolutionary pressure, represent a wide research field for scientists from different disciplines and the study of the determinants of their stability has been an important task for basic and applied research. A surprising conclusion emerges from these studies: there are no general rules to achieve protein stabilization. Each extremophilic protein adopts various strategies and the outstanding adaptation to extreme temperature and solvent conditions is realized through the same weak electrostatic and hydrophobic interactions among the ordinary amino acid residues which are also responsible for the proper balance between protein stability and flexibility in mesophilic proteins.

Thermal unfolding and conformational stability of the recombinant domain II of glutamate dehydrogenase from the hyperthermophile Thermotoga maritima.

Domain II (residues 189-338, M(r) = 16 222) of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was used as a model system to study reversible unfolding thermodynamics of this hyperthermostable enzyme. The protein was produced in large quantities in E.COLI: using a T7 expression system. It was shown that the recombinant domain is monomeric in solution and that it comprises secondary structural elements similar to those observed in the crystal structure of the hexameric enzyme. The recombinant domain is thermostable and undergoes reversible and cooperative thermal unfolding in the pH range 5.90-8.00 with melting temperatures between 75.1 and 68.0 degrees C. Thermal unfolding of the protein was studied using differential scanning calorimetry and circular dichroism spectroscopy. Both methods yielded comparable values. The analysis revealed an unfolding enthalpy at 70 degrees C of 70.2 +/- 4.0 kcal/mol and a DeltaC(p) value of 1.4 +/- 0.3 kcal/mol K. Chemical unfolding of the recombinant domain resulted in m values of 3.36 +/- 0.10 kcal/mol M for unfolding in guanidinium chloride and 1.46 +/- 0.04 kcal/mol M in urea. The thermodynamic parameters for thermal and chemical unfolding equilibria indicate that domain II from T.MARITIMA: glutamate dehydrogenase is a thermostable protein with a DeltaG(max) of 3.70 kcal/mol. However, the thermal and chemical stabilities of the domain are lower than those of the hexameric protein, indicating that interdomain interactions must play a significant role in the stabilization of T. MARITIMA: domain II glutamate dehydrogenase.

Stability, refolding and Ca2+ binding of pullulanase from the hyperthermophilic archaeon Pyrococcus woesei.

The unfolding and refolding of the extremely heat-stable pullulanase from Pyrococcus woesei has been investigated using guanidinium chloride as denaturant. The monomeric enzyme (90 kDa) was found to be very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 4.86 +/- 0.29 M for intrinsic fluorescence and 4.90 +/- 0.31 M for far-UV CD changes. The unfolding process was reversible. Reactivation of the completely denatured enzyme (in 7.8 M guanidinium chloride) was obtained upon removal of the denaturant by stepwise dilution; 100% reactivation was observed when refolding was carried out via a guanidinium chloride concentration of 4 M in the first dilution step. Particular attention has been paid to the role of Ca2+ which activates and stabilizes this archaeal pullulanase against thermal inactivation. The enzyme binds two Ca2+ ions with a Kd of 0.080 +/- 0.010 microM and a Hill coefficient H of 1.00 +/- 0.10. This cation enhances significantly the stability of the pullulanase against guanidinium chloride-induced unfolding and the DeltaGH2OD increased from 6.83 +/- 0.43 to 8.42 +/- 0.55 kcal.mol-1. The refolding of the pullulanase, on the other hand, was not affected by Ca2+.

A single-chain antibody fragment is functionally expressed in the cytoplasm of both Escherichia coli and transgenic plants.

Despite the well-known crucial role of intradomain disulfide bridges for immunoglobulin folding and stability, the single-chain variable fragment of the anti-viral antibody F8 is functionally expressed when targeted to the reducing environment of the plant cytoplasm. We show here that this antibody fragment is also functionally expressed in the cytoplasm of Escherichia coli. A gel shift assay revealed that the single-chain variable fragment (scFv) accumulating in the plant and bacterial cytoplasm bears free sulfhydryl groups. Guanidinium chloride denaturation/renaturation studies indicated that refolding occurs even in a reducing environment, producing a functional molecule with the same spectral properties of the native scFv(F8). Taken together, these results suggest that folding and functionality of this antibody fragment are not prevented in a reducing environment. This antibody fragment could therefore represent a suitable framework for engineering recombinant antibodies to be targeted to the cytoplasm.

A monomeric mutant of Clostridium symbiosum glutamate dehydrogenase: comparison with a structured monomeric intermediate obtained during refolding.

The refolding of Clostridium symbiosum glutamate dehydrogenase (GDH) involves the formation of an inactive structured monomeric intermediate prior to its concentration-dependent association. The structured monomer obtained after removal of guanidinium chloride was stable and competent for reconstitution into active hexamers. Site-directed mutagenesis of C. symbiosum gdh gene was performed to replace the residues Arg-61 and Phe-187 which are involved in subunit-subunit interactions, as determined by three-dimensional structure analysis. Heterologous over-expression in Escherichia coli of the double mutant (R61E/F187D) led to the production of a soluble protein with a molecular mass consistent with the monomeric form of clostridial GDH. This protein is catalytically inactive but cross-reacts with an anti-wild-type GDH antibody preparation. The double mutant R61E/F187D does not assemble into hexamers. The physical properties and the stability toward guanidinium chloride and urea of R61E/F187D were studied and compared to those of the structured monomeric intermediate.

Protein thermostability in extremophiles.

Thermostability of a protein is a property which cannot be attributed to the presence of a particular amino acid or to a post synthetic modification. Thermostability seems to be a property acquired by a protein through many small structural modifications obtained with the exchange of some amino acids and the modulation of the canonical forces found in all proteins such as electrostatic (hydrogen bonds and ion-pairs) and hydrophobic interactions. Proteins produced by thermo and hyperthermophilic microorganisms, growing between 45 and 110 degrees C are in general more resistant to thermal and chemical denaturation than their mesophilic counterparts. The observed structural resistance may reflect a restriction on the flexibility of these proteins, which, while allowing them to be functionally competent at elevated temperatures, renders them unusually rigid at mesophilic temperatures (10-45 degrees C). The increased rigidity at mesophilic temperatures may find a structural determinant in increased compactness. In thermophilic proteins a number of amino acids are often exchanged. These exchanges with some strategic placement of proline in beta-turns give rise to a stabilization of the protein. Mutagenesis experiments have confirmed this statement. From the comparative analysis of the X-ray structures available for several families of proteins, including at least one thermophilic structure in each case, it appears that thermal stabilization is accompanied by an increase in hydrogen bonds and salt bridges. Thermostability appears also related to a better packing within buried regions. Despite these generalisations, no universal rules can be found in these proteins to achieve thermostability.

Acid-induced disassembly of glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus occurs below pH 2.0.

The stability of the hexameric glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus at low pH values has been studied by activity assay, spectroscopic methods, size-exclusion chromatography and ultracentrifugation analysis. The enzyme is exceptionally stable and at pH 2.0 its hexameric assembly is preserved despite the changes observed in its tertiary structure. Below pH 1.7 dissociation into monomers starts and is accompanied by a progressive loss of tertiary interactions. Dissociation intermediate(s) were not detectable. At pH 2.0 the addition of NaCl causes the same structural changes observed upon further addition of protons. The monomeric state of the enzyme at pH 1.0 shows a significant content of native secondary structure and can be unfolded by guanidinium chloride. The role of electrostatic interactions in the high stability of the enzyme structure at low pH values is discussed.

Refolding pathway and association intermediates of glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus.

The denaturation and renaturation processes of the hexameric glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus have been investigated using guanidinium chloride as denaturant. The enzyme is highly stable and the transition midpoint for guanidinium chloride denaturation is 6.1 M. The recovery of enzyme structure occurs after dilution of the denaturant at 20 degrees C through the formation of structured monomers. Concentration of the structured monomers leads to the formation of higher association states with a tertiary structure different from that of the native enzyme. Activity is observed only in the presence of the hexamers, although a heating step at 70 degrees C is required to fully reactivate the hexamer formed at 20 degrees C. The refolding process and the intermediate(s) were studied by activity assay, spectroscopic methods, size-exclusion chromatography, and ultracentrifugation analysis.

Exchange of domains of glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus and the mesophilic bacterium Clostridium difficile: effects on catalysis, thermoactivity and stability.

The glutamate dehydrogenase gene from the hyperthermophilic archaeon Pyrococcus furiosus has been functionally expressed in Escherichia coli under the control of the lambda PL promoter. The P. furiosus glutamate dehydrogenase amounted to 20% of the total E. coli cell protein, and the vast majority consisted of hexamers. Following activation by heat treatment, an enzyme could be purified from E. coli that was indistinguishable from the glutamate dehydrogenase purified from P. furiosus. Hybrid genes, that consisted of the coding regions for the homologous glutamate dehydrogenases from P. furiosus and the mesophilic bacterium Clostridium difficile, were constructed and successfully expressed in E. coli. One of the resulting hybrid proteins, containing the glutamate binding domain of the C. difficile enzyme and the cofactor binding domain of the P. furiosus enzyme, did not show a detectable activity. In contrast, the complementary hybrid containing the P. furiosus glutamate and the C. difficile cofactor binding domain was a catalytically active hexamer that showed a reduced substrate affinity but maintained efficient cofactor binding with the specificity found in the Clostridium symbiosum enzyme. Compared with the C. difficile glutamate dehydrogenase, the archaeal-bacterial hybrid is slightly more thermoactive, less thermostable but much more stable towards guanidinium chloride-induced inactivation and denaturation.

The structure of Pyrococcus furiosus glutamate dehydrogenase reveals a key role for ion-pair networks in maintaining enzyme stability at extreme temperatures.

BACKGROUND: The hyperthermophile Pyrococcus furiosus is one of the most thermostable organisms known, with an optimum growth temperature of 100 degrees C. The proteins from this organism display extreme thermostability. We have undertaken the structure determination of glutamate dehydrogenase from P. furiosus in order to gain further insights into the relationship between molecular structure and thermal stability. RESULTS: The structure of P. furiosus glutamate dehydrogenase, a homohexameric enzyme, has been determined at 2.2 A resolution and compared with the structure of glutamate dehydrogenase from the mesophile Clostridium symbiosum. CONCLUSIONS: Comparison of the structures of these two enzymes has revealed one major difference: the structure of the hyperthermophilic enzyme contains a striking series of ion-pair networks on the surface of the protein subunits and buried at both interdomain and intersubunit interfaces. We propose that the formation of such extended networks may represent a major stabilizing feature associated with the adaptation of enzymes to extreme temperatures.

Crystallization of the NAD(P)-dependent glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus.

The NAD(P)-dependent glutamate dehydrogenase from Pyrococcus furiosus has been crystallized by the hanging-drop method of vapour diffusion using lithium sulfate as the precipitant. The crystals belong to the tetragonal system and are in space group P4(2)2(1)2 with unit-cell dimensions of a = b = 167.2, c = 172.9 A. Consideration of the values of V(m) and possible packing of the molecules within the cell suggest that the asymmetric unit contains a trimer. P. furiosus belongs to the family of Archaea and is one of the most thermostable organisms known, having an optimal growth temperature of 376 K. The glutamate dehydrogenase isolated from this organism has a half-life of 12 h at 373 K and, therefore, the determination of the structure of this enzyme will be important in advancing our understanding of how proteins are adapted to enable them to survive at such extreme temperatures.

Refolding of glutamate dehydrogenase from Bacillus acidocaldarius after guanidinium chloride-induced unfolding.

The hexameric NAD-dependent glutamate dehydrogenase from the thermophilic eubacterium Bacillus acidocaldarius is the first glutamate dehydrogenase which spontaneously refolds in vitro. After 24 h unfolding in 6 M guanidinium chloride at 20 degrees C, refolding was achieved by dilution of the denaturant. The yield of reconstitution obtained in the presence of 1,4 dithio-DL-threitol in the unfolding/refolding mixture was 75%. The unfolding/refolding equilibria have been studied by fluorescence, circular dichroism and catalytic activity, which was 50% inhibited at 0.08 M guanidinium chloride, a value 30-fold lower than the transition midpoint detected by physical changes. Refolding was attempted in the presence of various additives, at different temperatures and varying enzyme and residual guanidinium chloride concentration. The refolded enzyme, after removal of inactive aggregated species, completely resembles the native enzyme in term of its physicochemical and kinetic properties as well as thermophilicity.

The amino acid sequence of glutamate dehydrogenase from Pyrococcus furiosus, a hyperthermophilic archaebacterium.

The complete amino acid sequence of glutamate dehydrogenase from the archaebacterium Pyrococcus furiosus has been determined. The sequence was reconstructed by automated sequence analysis of peptides obtained after cleavage with cyanogen bromide, Asp-N endoproteinase, trypsin, or pepsin. The enzyme subunit is composed of 420 amino acid residues yielding a molecular mass of 47,122 D. In the recently determined primary structure of glutamate dehydrogenase from another thermophilic archaebacterium, Sulfolobus solfataricus, the presence of some methylated lysines was detected and the possible role of this posttranslational modification in enhancing the thermostability of the enzyme was discussed (Maras, B., Consalvi, V., Chiaraluce, R., Politi, L., De Rosa, M., Bossa, F., Scandurra, R., and Barra, D. (1992), Eur. J. Biochem. 203, 81-87). In the primary structure reported here, such posttranslational modification has not been found, indicating that the role of lysine methylation should be revisited. Comparison of the sequence of glutamate dehydrogenase from Pyrococcus furiosus with that of S. solfataricus shows a 43.7% similarity, thus indicating a common evolutionary pathway.

Glutamate dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus: studies on thermal and guanidine-dependent inactivation.

The hexameric NAD(P)-dependent glutamate dehydrogenase isolated from the thermoacidophilic archaebacterium Sulfolobus solfataricus shows a remarkable thermal stability which is strictly dependent on protein concentration (half-life at 95 degrees C is 0.25 h and 0.5 h at 0.4 and 0.8 mg/ml, respectively). Temperature-dependent inactivation of the enzyme is apparently irreversible; this process is accompanied by a progressive increase in hydrophobic surface area which leads to protein precipitation. 3 M GdnHCl increases the half-life of the enzyme at 90 degrees C and 0.2 mg/ml 6-fold. The hexamer is the only soluble molecular species revealed by glutaraldehyde fixation after thermal inactivation. Lyotropic salts strongly affect the enzyme thermal stability: the half-life at 90 degrees C and 0.2 mg/ml protein concentration increases more than 6-fold in the presence of 0.4 M Na2SO4 and decreases 4-fold in the presence of 0.4 M NaSCN. The maximum protein thermal stability is observed around the isoelectric pH, between pH 5.2 and pH 6.8. Guanidine-dependent inactivation of the enzyme at 20 degrees C is irreversible above 1.5 M GdnHCl. The decline in percentage of reactivation closely parallels the structural changes detected by fluorescence and the loss of hexameric structure accompanied by the dissociation to monomers, as indicated by glutaraldehyde fixation.