Biomedical subjects

H Bedouelle

Publications and source records attributed to H Bedouelle.

At least 19 recordsLinked to original sources

PubMed2001

An essential residue in the flexible peptide linking the two idiosynchratic domains of bacterial tyrosyl-tRNA synthetases.

Tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus comprises three sequential domains: an N-terminal catalytic domain, an alpha-helical domain with unknown function, and a C-terminal tRNA binding domain (residues 320-419). The properties of the polypeptide segment that links the alpha-helical and C-terminal domains, were analyzed by measuring the effects of sequence changes on the aminoacylation of tRNA(Tyr) with tyrosine. Mutations F323A (Phe323 into Ala), S324A, and G325A showed that the side chain of Phe323 was essential but not those of Ser324 and Gly325. Insertions of Gly residues between Leu322 and Phe323 and the point mutation L322P showed that the position and precise orientation of Phe323 relative to the alpha-helical domain were important. Insertions of Gly residues between Gly325 and Asp326 and deletion of residues 330-339 showed that the length and flexibility of the sequence downstream from Gly325 were unimportant but that this sequence could not be deleted. Mutations F323A, -L, -Y, and -W showed that the essential property of Phe323 was its aromaticity. The Phe323 side chain contributed to the stability of the initial complex between TyrRS and tRNA(Tyr) for 2.0 +/- 0.2 kcal x mol(-1) and to the stability of their transition state complex for 4.2 +/- 0.1 kcal x mol(-1), even though it is located far from the catalytic site. The results indicate that the disorder of the C-terminal domain in the crystals of TyrRS is due to the flexibility of the peptide that links it to the helical domain. They identified Phe323 as an essential residue for the recognition of tRNA(Tyr).

Alanine↗

PubMed2000

The anticodon-binding domain of tyrosyl-tRNA synthetase: state of folding and origin of the crystallographic disorder.

The C-terminal domain (residues 320-419) of tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus is disordered in the crystal structure. Its function consists of binding the anticodon of tRNA(Tyr). We undertook to characterize its conformational state. A hybrid between the C-terminal fragment and a His-tag sequence was constructed and purified in large amounts. Analyses by mass spectrometry and analytical ultracentrifugation showed that the C-terminal fragment, thus purified, was not degraded and that it neither dimerized nor aggregated. Its far- and near-UV circular dichroism spectra revealed a high content in secondary structures and an asymmetrical environment of its aromatic residues. Each spectrum could be reconstructed by the difference between the corresponding spectra for the full-length TyrRS and for its N-terminal fragment. The Stokes radius of the C-terminal fragment, measured by size exclusion chromatography, indicated a condensed globular state. The fluorescence of ANS (a small hydrophobic probe) showed that the surface of the C-terminal fragment was more hydrophilic than that of a molten globule. These results on the C-terminal fragment and our previous observations that it can undergo cooperative transitions, demonstrated the following points: it is not in a disordered or molten globular state, it has a defined and stable three-dimensional structure, its structures are similar in its isolated and integrated forms, and the apparent disorder in the crystals of the full-length synthetase must be due to the flexibility of the polypeptide segment that links the N- and C-terminal domains. Thus, TyrRS has not evolved strong noncovalent interactions between its catalytic and anticodon-binding domains, contrary to the other synthetases.

Anilino Naphthalenesulfonates↗

PubMed2000

Mutational scanning of a hairpin loop in the tryptophan synthase beta-subunit implicated in allostery and substrate channeling.

The tryptophan synthases from Escherichia coli and Salmonella typhimurium are tetrameric enzymes, with an elongated TrpA.TrpB.TrpB.TrpA structure. Structural studies have identified residues 277-283 of TrpB as a potentially important region for the allosteric communication between the TrpA and TrpB subunits and for the transport of indole between their active sites through a hydrophobic tunnel. To explore the functional role of this region, we analyzed the effects of 19 single and double mutations in TrpB on the tryptophan synthase (TSase) and serine deaminase (SDase) activities of the TrpB2 dimer, either in the presence or in the absence of the TrpA subunit. The mutations of residues 273-283 could be divided into 4 classes. Mutations 1278A, F280G and M282A decreased the SDase and TSase activities of TrpB2 to similar extents. F280A decreased the SDase activity of TrpB2 more than its TSase activity, whereas the reverse was true for Y279L. F280A decreased the activation factor of TrpB2 by TrpA, whereas F280G increased it. The reaction steps and intramolecular contacts that could be affected by the mutations are described. The sequence 278-IYFGM-282, which is present in E. coli and S. typhimurium, is only found in 5 out of 42 organisms, whereas the sequence VLHGX is found in 21 organisms. Our results identified several mutations that could be used as structural probes to analyze precisely the roles of residues 278-282 and their evolution.

Allosteric Site↗

PubMed1999

Secondary structure of the C-terminal domain of the tyrosyl-transfer RNA synthetase from Bacillus stearothermophilus: a novel type of anticodon binding domain?

The tyrosyl-tRNA synthetase catalyzes the activation of tyrosine and its coupling to the cognate tRNA. The enzyme is made of two domains: an N-terminal catalytic domain and a C-terminal domain that is necessary for tRNA binding and for which it was not possible to determine the structure by X-ray crystallography. We determined the secondary structure of the C-terminal domain of the tyrosyl-tRNA synthetase from Bacillus stearothermophilus by nuclear magnetic resonance methods and found that it is of the alpha+beta type. Its arrangement differs from those of the other anticodon binding domains whose structure is known. We also found that the isolated C-terminal domain behaves as a folded globular protein, and we suggest the presence of a flexible linker between the two domains.

Amino Acid Sequence↗

PubMed1999

Experimental evolution of a dense cluster of residues in tyrosyl-tRNA synthetase: quantitative effects on activity, stability and dimerization.

A dense cluster of eight residues was identified at the crossing of two alpha-helices in tyrosyl-tRNA synthetase (TyrRS) from the thermophile Bacillus stearothermophilus. Its mechanism of evolution was characterized. Four residues of this cluster are not conserved in TyrRS from the mesophile Escherichia coli. The corresponding mutations were constructed in TyrRS(Delta1), a derivative of TyrRS from B. stearothermophilus in which the anticodon binding domain is deleted. Mutations I52L (i.e. Ile52 into Leu), M55L and L105V did not affect the activity of TyrRS(Delta1) in the pyrophosphate exchange reaction whereas T51P increased it. The kinetic stabilities of TyrRS(Delta1) and its mutant derivatives at 68.5 degreesC were determined from experiments of irreversible thermal precipitation. They were in the order L105V<I52L<T51P<Wild Type</=M55L; mutation I52L partially compensated L105V in these experiments whereas M55L was coupled neither to I52L nor to L105V. Mutations I52L and L105V affected the stability of the dimeric TyrRS(Delta1) at different steps of its unfolding by urea, monitored under equilibrium conditions by spectrofluorometry or size exclusion chromatography. I52L destabilized the association between the subunits even though residue Ile52 is more than 20 A away from the subunit interface. L105V destabilized the monomeric intermediate of unfolding. The two mutational pathways, going from the wild-type TyrRS(Delta1) to the I52L-L105V double mutant through each of the single mutants were not equivalent for the stability of the monomeric intermediate and for the total stability of the dimer. One pathway contained two neutral steps whereas the other pathway contained a destabilizing step followed by a stabilizing step. Mutation I52L allowed L105V along the first pathway and compensated it along the second pathway. Thus, the effects of I52L and L105V on stability depended on the structural context. The gain in activity due to T51P was at the expense of a slight destabilization.

Amino Acid Substitution↗

PubMed1999

Functional characterization of the somatic hypermutation process leading to antibody D1.3, a high affinity antibody directed against lysozyme.

The impact of somatic hypermutation on the affinity of Abs directed against protein Ags remains poorly understood. We chose as a model the secondary response Ab D1.3 directed against hen egg lysozyme. During the maturation process leading to this Ab, five replacement somatic mutations occurred. After reconstituting the germline Ab from which D1.3 originated, we assessed the energetic and kinetic importance of each of the somatic mutations, individually or combined, using the BIAcore apparatus. We found that the mutations induced an overall 60-fold improvement of affinity, principally due to a decrease in the kinetic rate of dissociation. We showed that their effects were additive and context independent; therefore, in the case of D1.3, the order in which somatic mutations were introduced and selected is unimportant. Interestingly, most of the affinity improvement was due to a single somatic mutation (Asn50-->Tyr in VL), involving a residue that belongs to the functional interface between Ab D1.3 and lysozyme. This replacement could either establish new Van der Waals contacts between the Ab and the Ag or help stabilize the conformation of a closely situated crucial residue of the Ab paratope. The four other mutations played only a marginal part in affinity maturation; potential reasons for which these mutations were nevertheless selected are discussed.

Animals↗

PubMed1999

Disordered C-terminal domain of tyrosyl-tRNA synthetase: secondary structure prediction.

The C-terminal domain (residues 320-419) of tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus is disordered in the crystal structure and involved in the binding of the anticodon arm of tRNA(Tyr). The sequences of 11 TyrRSs of prokaryotic or mitochondrial origins were aligned and the alignment showed the existence of conserved residues in the sequences of the C-terminal domains. A consensus could be deduced from the application of five programs of secondary structure prediction to the 11 sequences of the query set. These results suggested that the sequences of the C-terminal domains determined a precise and conserved secondary structure. They predicted that the C-terminal domain would have a mixed fold (alpha/beta or alpha+beta), with the alpha-helices in the first half of the sequence and the beta-strands mainly in its second half. Several programs of fold recognition from sequence alone, by threading onto known structures, were applied but none of them identified a type of fold that would be common to the different sequences of the query set. Therefore, the fold of the C-terminal, anticodon binding domain might be novel.

Amino Acid Sequence↗

PubMed1998

A mutational approach shows similar mechanisms of recognition for the isolated and integrated versions of a protein epitope.

Antibody mAb164 is directed against the native form of the TrpB2 subunit of Escherichia coli tryptophan synthase. It recognizes a synthetic peptide, P11, constituted of residues 273-283 of TrpB, with high affinity. We introduced 16 single and 3 double mutations in each of the two contexts, TrpB2 and P11, and used them as local probes to study the cross-reactivity of mAb164 toward these two antigens. The equilibrium constant, KD, of dissociation from mAb164 was measured for each of the mutant derivatives of TrpB2 and P11 by a competition enzyme-linked immunosorbent assay and compared with the wild type one. The variation of the free energy of interaction, DeltaDeltaG, covered nearly 8 kcal/mol for the different mutations. The values of DeltaDeltaG for the mutant derivatives of TrpB2 and for those of P11 were close and the two sets of values were strongly correlated (r = 0.96). This correlation showed that mAb164 recognized the integrated and isolated versions of residues 273-283 with very similar mechanisms. A few significant differences between the recognitions of TrpB2 and P11 by mAb164 suggested some adaptability of the interaction. The results were compatible with a recognition of residues 273-283 of TrpB in a loop conformation, close to their structure in the crystals of the complete tryptophan synthase, TrpA2TrpB2.

Antibodies, Bacterial↗

PubMed1998

The rate of dissociation between antibody and antigen determines the efficiency of antibody-mediated antigen presentation to T cells.

We analyzed the role of Ab affinity on Ab-mediated Ag uptake and presentation to T cells. Hen egg white lysozyme (HEL) was captured by bifunctional hybrid proteins (Fv-MalE) in which the variable fragment (Fv) of the anti-HEL mAb D1.3 was covalently linked to the Escherichia coli MalE protein. These complexes were targeted via two anti-MalE mAbs to an APC expressing a receptor for the Ab constant region. The combination of Fv-MalE and anti-MalE mAbs increased, specifically, HEL presentation. With this experimental system, we evaluated the impact of six different mutations, affecting the Fv-MalE complementarity determining regions, on the increase of HEL presentation by the corresponding hybrids. These mutations increase the dissociation rate constant (k(off)), and, thus, the dissociation constant of the HEL/Fv-MalE interaction, up to 650-fold, as compared with the wt Fv-MalE. Increasing the k(off) from 7 x 10(-4) s(-1) up to 300 x 10(-4) s(-1) did not interfere with the enhancement of HEL presentation. A mutant with a k(off) of 600 x 10(-4) s(-1) had a reduced enhancement ability, and mutants with k(off) higher than 5700 x 10(-4) s(-1) did not enhance HEL presentation at all. These results show that affinity determines the efficiency of Ab-mediated Ag presentation to T cells. One consequence is that affinity maturation in specific B lymphocytes can drastically enhance their ability to collaborate with T cells in an MHC-restricted way. This may contribute to the selection of high affinity B cell clones.

ATP-Binding Cassette Transporters↗

PubMed1998

Dimeric tyrosyl-tRNA synthetase from Bacillus stearothermophilus unfolds through a monomeric intermediate. A quantitative analysis under equilibrium conditions.

Tyrosyl-tRNA synthetase from Bacillus stearothermophilus comprises an N-terminal domain (residues 1-319), which is dimeric and forms tyrosyladenylate, and a C-terminal domain (residues 320-419), which binds the anticodon arm of tRNATyr. The N-terminal domain has the characteristic fold of the class I aminoacyl-tRNA synthetases. The unfolding of the N-terminal domain by urea at 25 degreesC under equilibrium conditions was monitored by its intensities of light emission at 330 and 350 nm, the ratio of these intensities, its ellipticity at 229 nm, and its partition coefficient, in spectrofluorometry, circular dichroism, and size-exclusion chromatography experiments, respectively. These experiments showed the existence of an equilibrium between the native dimeric state of the N-terminal domain, a monomeric intermediate state, and the unfolded state. The intermediate was compact and had secondary structure, and its tryptophan residues were partially buried. These properties of the intermediate and its inability to bind 1-anilino-8-naphthalenesulfonate showed that it was not in a molten globular state. The variation of free energy deltaG(H2O) and its coefficient m of dependence on the concentration of urea were, respectively, 13.8 +/- 0.2 kcal.mol-1 and 0.9 +/- 0.1 kcal.mol-1.M-1 for the dissociation of the native dimer and 13.9 +/- 0.6 kcal.mol-1 and 2.5 +/- 0.1 kcal.mol-1.M-1 for the unfolding of the monomeric intermediate.

Chromatography, Gel↗

PubMed1997

Conformational and functional properties of an undecapeptide epitope fused with the C-terminal end of the maltose binding protein.

Monoclonal antibody mAb164 is directed against the TrpB2 subunit of the Escherichia coli tryptophan synthase. It recognizes the synthetic peptide P11, constituted of residues 273-283 of TrpB, with high affinity. We constructed a hybrid protein in which the C-terminal end of protein MalE was linked with the N-terminal end of P11. Hybrid MalE-P11 was produced in E. coli from a plasmidic gene and purified in one step as MalE. MalE-P11 and the isolated P11 had identical conformational and functional properties according to the following criteria. The NMR spectra of MalE and MalE-P11 in TOCSY experiments showed that the P11 moiety of MalE-P11 moved independently from its MalE moiety. The chemical shifts of the protons for the P11 moiety of MalE-P11 and for the isolated P11 were very close and did not show significant deviations from random coil values. The equilibrium constant of dissociation (KD) from mAb164, measured by a competition ELISA, was identical for MalE-P11 and the isolated P11, around 6 nM. The change of the C-terminal residue of MalE-P11 from Lys into Ala increased 37-fold this dissociation constant. This increase showed that the P11 moiety of MalE-P11 was not degraded. The high molecular mass of MalE-P11 allowed us to follow its kinetics of interaction with immobilized mAb164 by surface plasmon resonance, using the BIAcore apparatus. The rates of association with mAb164 were similar for MalE-P11 and TrpB2, but the dissociation was faster for MalE-P11 than for TrpB2, as previously observed for the isolated P11 by a fluorometric method. Thus, the fusion of peptides with the C-terminal end of MalE could constitute an alternative to chemical synthesis for the study of their recognition by receptors, in vivo or in vitro.

ATP-Binding Cassette Transporters↗

PubMed1997

Mutational analysis of an antigenic peptide shows recognition in a loop conformation.

We have analyzed the recognition between an antigenic undecapeptide and a monoclonal antibody through a mutational approach. Antibody mAb164 is directed against the native form of the TrpB2 subunit of Escherichia coli tryptophan synthase. It recognizes a synthetic peptide, P11, constituted of residues 273-HGRVGIYFGMK-283 of TrpB with high affinity. P11 was fused with a carrier protein, MalE, to facilitate its manipulation. The affinities between mAb164 and the MalE-P11 hybrids were measured by competition enzyme-linked immunosorbent assay (ELISA). The changes of the P11 residues into progressively shorter residues, the comparison of changes into Pro and Ala, and the study of double mutants showed the following. Four hydrophobic residues of P11, Val276, Ile278, Tyr279, and Phe280, were predominant in the interaction. For some residues, e.g., Tyr279, most groups of the side chain contributed to the interaction. For others, only some groups played a significant role, e.g., the Cdelta group of Ile278 or the Cbeta group of Phe280. The lack of side chain in position Gly281 and a tertiary interaction between the side chains of Ile278 and Lys283 were important. P11 was recognized in a loop conformation, close to that of residues 273-283 of TrpB in the crystal structure of the complete tryptophan synthase, TrpA2TrpB2. Comparison of our mutational data with NMR data on the conformation of the isolated peptide P11 and with kinetic data on its interaction with mAb164 indicate that mAb164 selects a conformer of P11 that represents only a small minority of the molecules. Our results provide useful information on the mechanisms by which linear epitopes and unconstrained peptides are recognized by receptors.

ATP-Binding Cassette Transporters↗

PubMed1997

Energetic and kinetic contributions of contact residues of antibody D1.3 in the interaction with lysozyme.

Fully functional variable fragments (Fv) of D1.3, a mouse antibody directed against the hen egg lysozyme, were readily produced as hybrids (Fv-MalE) with the maltose-binding protein of Escherichia coli and purified independently of their antigen-binding properties. We used site-directed mutations of residues in the complementarity-determining regions (CDRs) of D1.3 as local conformational probes, and compared their effects on the binding of Fv and Fv-MalE to lysozyme. We found that the MalE moiety did not significantly interfere with the interaction between the antigen and the antibody Fv fragment. We then determined the contribution of several potential contact residues of D1.3 in the interaction with lysozyme, by assaying the effect of site-directed mutations on the kinetics of association and dissociation of the complex between Fv-MalE and immobilized lysozyme, using the BIAcore apparatus. While the k(on) values were virtually unaffected by the mutations, the k(off) values varied by more than three orders of magnitude. Both charged (aspartate and arginine) and aromatic (tyrosine and tryptophan) residues in the CDR3 regions of the heavy and light chains of D1.3, which form the center of its antigen-combining site, played a preponderant part in the binding of lysozyme. Our results also showed that indirect hydrogen bonds, bridged by water molecules, contributed significantly to the interaction between D1.3 and lysozyme, and that their energy could be estimated at 1 to 2 kcal.mol-1.

ATP-Binding Cassette Transporters↗

PubMed1996

Disordered C-terminal domain of tyrosyl transfer-RNA synthetase: evidence for a folded state.

The C-terminal domain (residues 320 to 419) of tyrosyl-tRNA synthetase from Bacillus stearothermophilus (Bst-TyrRS) is necessary for the binding of tRNA(Tyr) but disordered in the crystal structure. Four different criteria showed that the isolated C-terminal domain of Bst-TyrRS was at least partially folded in solution. Its spectrum of circular dichroism was compatible with a high content of secondary structure elements (56% of its residues) and these structural elements disappeared in 7.5 M urea. It was unfolded by urea along a unique transition, around 6.0 M, according to the variations in the fluorescence of its tyrosine residues and in its electrophoretic mobility through transverse gradient gels. It was denatured by heat with a temperature of half-precipitation in 30 minutes that was equal to 67.9 degrees C and close to the Bst-TyrRS one, 68.7 degrees C. Its transitions of denaturation by urea or temperature were weakly cooperative. The C-terminal domains of the TyrRSs from Escherichia coli (Eco-TyrRS) and B. stearothermophilus could be genetically exchanged without a significant loss of aminoacylation activity. A hybrid between the N-terminal domain of Bst-TyrRS and the C-terminal domain of Eco-TyrRS was precipitated by heat in 30 minutes following two transitions: 83% of the molecules were precipitated with a temperature of half-transition (51.6 degrees C) close to the Eco-TyrRS one (48.6 degrees C). The remainder was precipitated with a temperature of half-transition (65.5 degrees C) close to the Bst-TyrRS one (67.2 degrees C) or that of its N-terminal domain (68.0 degrees C). These results showed that the C-terminal domain of Eco-TyrRS could undergo a transition from a soluble active conformation to an insoluble one. The denaturations of Bst-TyrRS and of its N-terminal domain by urea occurred with two successive transitions, around 4 M and 6 M, and thus according to a complex mechanism.

Base Sequence↗

PubMed1996

Destabilizing interactions between the partners of a bifunctional fusion protein.

Hybrid MalE-GVP is a bifunctional protein in vitro since it binds maltose as protein MalE of Escherichia coli and since it is dimeric and specifically binds single-stranded DNA as protein GVP of phage M13. The oxidation rate of a unique cysteine residue was used to compare the stabilities of GVP in its free and hybrid forms, under conditions where MalE was either folded or unfolded by a denaturing agent. The results showed that both the covalent link and tertiary non-covalent interactions between MalE and GVP destabilized GVP in MalE-GVP. To test whether GVP had identical structures in its free and hybrid forms, mutations were used as local conformational probes. The effects of these mutations on the capabilities of MalE-GVP to dimerize and to bind single-stranded DNA were assayed in vitro. They were compatible with the effects of the same mutations on the global activity of free GVP in vivo and with the effects that could be predicted from the known data on free GVP, in particular its crystal structure. Thus, one partner of a hybrid protein can be destabilized by the other partner while maintaining its structural and functional characteristics.

ATP-Binding Cassette Transporters↗

PubMed1995

Macromolecular recognition through electrostatic repulsion.

In the process of genetic translation, each aminoacyl-tRNA synthetase specifically aminoacylates its cognate tRNAs and rejects the 19 other species of tRNAs. A decrease in the specificity of this reaction can result in misincorporations of amino acids into proteins and be deleterious to the cell. In the case of tyrosyl-tRNA synthetase from Bacillus stearothermophilus, the change of residue Glu152 into Ala results in erroneous interactions with non-cognate tRNAs. To analyse how Glu152 contributes to the discrimination between tRNAs by tyrosyl-tRNA synthetase, 11 changes to this residue were created by mutagenesis. The misaminoacylations of tRNA(Phe) and tRNA(Val) with tyrosine in vitro (on a scale going from 1 to 30) and the toxicity of tyrosyl-tRNA synthetase in vivo (on a scale from 1 to 10(7)) increased in a correlated way when the nature of the side chain in position 152 varied from negatively charged to uncharged then to positively charged. The aminoacylation of tRNA(Tyr) was unaffected by the mutations. The results show that the role of Glu152 in the discrimination between tRNAs is purely negative, that it acts by electrostatic repulsion of non-cognate tRNAs and that this mechanism has been conserved throughout evolution.

Biological Evolution↗

PubMed1994

Bifunctional hybrids between the variable domains of an immunoglobulin and the maltose-binding protein of Escherichia coli: production, purification and antigen binding.

Hybrids were constructed between the maltose-binding protein of Escherichia coli (MalE) and the variable domains (V-domains) of D1.3, a mouse antibody directed against hen lysozyme. Each V-domain was fused with the C- or N-terminus of MalE and expressed in E. coli, either alone or associated with the other V-domain, as a heterodimer (Fv) or as a single-chain fragment (scFv). The hybrids were exported into the bacterial periplasm, purified by affinity chromatography on cross-linked amylose and separated from incomplete products by ion-exchange chromatography. Hybrids between MalE and Fv bound the antigen specifically, with affinities increased up to 10-fold when compared to native D1.3. This strongly suggests that MalE contributed to the binding. The affinities and specificities of the different hybrids, as well as their levels of contamination by incomplete products, depended on their fusion pattern with MalE. Hybrids between MalE and either single V-domain also bound hen lysozyme specifically, which shows that each V-domain can recognize the antigen when fused with MalE. The high affinity of VH-MalE (KD = 3 nM) could be due to both participation of MalE in the binding and a conformational adaptation of the lone V-domain.

ATP-Binding Cassette Transporters↗

PubMed1993

Mapping the stability determinants of bacterial tyrosyl transfer RNA synthetases by an experimental evolutionary approach.

The tyrosyl-tRNA synthetases from Bacillus stearothermophilus (Bst-TyrTS) and Escherichia coli (Eco-TyrTS) are 56% identical in amino acid sequence. To map and characterize the set of interactions that makes Bst-TyrTS more stable than Eco-TyrTS, a family of nine hybrid proteins was constructed between the two enzymes. The N-terminal part of each hybrid came from Eco-TyrTS and the C-terminal part from Bst-TyrTS. The stability and activity of these hybrids were estimated by experiments of thermal inactivation and tRNA charging. For all the hybrids, the temperature of half-inactivation in 30 minutes was above 44 degrees C and the rate of charging was at least 40% that of Bst-TyrTS. In general, the temperature of half-inactivation increased and the rate of charging decreased monotonically when the number of residues coming from the more stable and less active Bst-TyrTS increased. As a result, the rate of charging decreased when the temperature of half-inactivation increased. These results show that the sequences and structures of the two enzymes can replace each other locally and still give a stable and active TyrTS, and that the greater stability of Bst-TyrTS is due to cumulative changes of residues scattered along the sequence. They suggest that Bst-TyrTS is more rigid than Eco-TyrTS at low temperature. The existence of a few exceptional hybrids, having stabilities or activities lower than those of the neighbouring hybrids, shows that compensatory changes of residues have occurred between the two sequences during evolution. These exceptions could be explained by the systematic identification of the couples of residues that are in contact in the Bst-TyrTS structure and become heterologous in some hybrids.

Binding Sites↗