Biomedical subjects

Juliette T J Lecomte

Publications and source records attributed to Juliette T J Lecomte.

At least 19 recordsLinked to original sources

PubMed2006

Structural and dynamic repercussions of heme binding and heme-protein cross-linking in Synechococcus sp. PCC 7002 hemoglobin.

The recombinant two-on-two hemoglobin from the cyanobacterium Synechoccocus sp. PCC 7002 (S7002 rHb) is a bishistidine hexacoordinate globin capable of forming a covalent cross-link between a heme vinyl and a histidine in the C-terminal helix (H helix). Of the two heme axial histidines, His46 (in the E helix, distal side) and His70 (in the F helix, proximal histidine), His46 is displaced by exogenous ligands. S7002 rHb can be readily prepared as an apoglobin (apo-rHb), a non-cross-linked hemichrome (ferric iron and histidine axial ligands, rHb-R), and a cross-linked hemichrome (rHb-A). To determine the effects of heme binding and subsequent cross-linking, apo-rHb, rHb-R, and rHb-A were subjected to thermal denaturation and 1H/2H exchange. Interpretation of the latter data was based on nuclear magnetic resonance assignments obtained with uniformly 15N- and 13C,15N-labeled proteins. Apo-rHb was found to contain a cooperative structural core, which was extended and stabilized by heme binding. Cross-linking resulted in further stabilization attributed mainly to an unfolded-state effect. Protection factors were higher at the cross-link site and near His70 in rHb-A than in rHb-R. In contrast, other regions became less resistant to exchange in rHb-A. These included portions of the B and E helices, which undergo large conformational changes upon exogenous ligand binding. Thus, the cross-link readjusted the dynamic properties of the heme pocket. 1H/2H exchange data also revealed that the B, G, and H helices formed a robust core regardless of the presence of the heme or cross-link. This motif likely encompasses the early folding nucleus of two-on-two globins.

Circular Dichroism↗

PubMed2006

Proximal influences in two-on-two globins: effect of the Ala69Ser replacement on Synechocystis sp. PCC 6803 hemoglobin.

The cyanobacterium Synechocystis sp. PCC 6803 (S6803) expresses a two-on-two globin in which His46 (distal side) and His70 (proximal) function as heme iron axial ligands. His46 can be displaced by O2, CO, and CN-, among others, whereas His70 is not labile under native conditions. The residue preceding the proximal histidine has been implicated in controlling globin axial ligand reactivity; the details of the mechanism, however, are not well understood, and little information exists for bis-histidyl hexacoordinate proteins. In many vertebrate hemoglobins and in the Synechocystis protein, the position is occupied by an alanine, whereas, in myoglobins, it is a serine involved in an intricate hydrogen-bond network. We examined the role of Ala69 in S6803 hemoglobin through the effects of an Ala --> Ser replacement. The substitution resulted in minor structural perturbations, but the response of the holoprotein to temperature-, urea-, and acid-induced denaturation was measurably affected. Enhanced three-state behavior was manifested in the decoupling of heme binding and secondary-structure formation. Urea-gradient gel experiments revealed that the stability of the apoprotein was unchanged by the replacement and that a slight alteration of the folding kinetics occurred in the holoproteins. Cyanide-binding experiments were performed to assess trans effects. The apparent rate constant for association decreased 2-fold upon Ala69Ser replacement. This deceleration was attributed to a change in the lifetime of a state containing a decoordinated His46. The results demonstrated that, as in vertebrate globins and leghemoglobin, proximal influences operate to determine fundamental dynamic and thermodynamic properties of the protein.

Alanine↗

PubMed2006

Cytosol has a small effect on protein backbone dynamics.

Cells are crowded with macromolecules, yet most biophysical information about proteins is obtained in dilute solution. To determine the impact of this dichotomy, we used nuclear magnetic resonance spectroscopy to measure the backbone (15)N T(1) and T(2) relaxation times and the {(1)H}-(15)N nuclear Overhauser enhancement (nOe) of uniformly (15)N-enriched apocytochrome b(5) in living Escherichia coli and in dilute solution. These data allowed us to assess the backbone dynamics of this partially folded protein in cells and in dilute solution. The two data sets were analyzed by using the model-free approach. Transfer from dilute solution to the cytosol has a quantitative effect on T(1), T(2), and nOe values. Most of the effects are attributed to an increase in the overall correlation time, caused by the increased viscosity of the cytosol compared to that of the dilute solution. Our main conclusion is that the cytosol does not alter the pattern of backbone dynamics of apocytochrome b(5). Increases in the time scale of both the picosecond and millisecond motions are observed, but the increases are less than approximately 30%.

Cell Nucleus↗

PubMed2006

A dynamic N-capping motif in cytochrome b5: evidence for a pH-controlled conformational switch.

Apocytochrome b5 is a marginally stable protein exhibiting under native conditions a slow conformational exchange in its C-terminal region. The affected elements of secondary structure include a 3(10)-helix containing at its N-terminus a histidine Ncap and a subsequent proline. Participation of the neutral histidine side-chain in backbone amide capping lowers the imidazole pKa. To explore the nature of the conformational exchange in the protein and determine whether it is related to cis-trans isomerization of the His-Pro bond, three octapeptides encompassing the helix were synthesized and studied by NMR spectroscopy. One corresponded to the wild-type sequence, the second was the D-histidine epimer, and the third contained an alanine in place of the proline. It was found that the rates of cis-trans interconversion in the proline-containing peptides were slower than the rates of the conformational exchange in the protein. In addition, the wild-type peptide hinted at a predisposition for Ncap formation when in the trans configuration. Analysis of the pH response of the peptides and protein suggested that at pH near neutral, the conformational exchange detected in the protein involved only species with a trans His-Pro bond and could be approximated with a three-state model by which the terminal helix sampled a locally unfolded state. This state, which contained an uncapped histidine with a normal pKa, partitioned into neutral and protonated populations according to pH. The intrinsic conformational bias of the wild-type peptide and the pH-driven equilibria illustrated how a 3(10)-element could serve as a nucleation site for structural rearrangement.

Animals↗

PubMed2006

A phylogenetic and structural analysis of truncated hemoglobins.

Truncated hemoglobins (trHbs) are heme proteins found in bacteria, plants, and unicellular eukaryotes. They are distantly related to vertebrate hemoglobins and are typically shorter than these by 20-40 residues. The multiple amino acid deletions, insertions, and replacements result in distinctive alterations of the canonical globin fold and a wide range of chemical properties. An early phylogenetic analysis categorized trHbs into three groups, I (trHbN), II (trHbO), and III (trHbP). Here, we revisit this analysis with 111 trHbs. We find that trHbs are orthologous within each group and paralogous across the groups. Group I globins form the most disparate set and separate into two divergent subgroups. Group II is comparatively homogeneous, whereas Group III displays the highest level of overall conservation. In Group I and Group II globins, for which some ligand binding and structural data are available, an improved description of probable protein-ligand interactions is achieved. Other conservation trends are either confirmed (essential glycines in loops), refined (lining of ligand access tunnel), or newly identified (helix start signal). The Group III globins, so far uncharacterized, exhibit recognizable heme cavity residues while lacking some of the residues thought to be important to the trHb fold. An analysis of the phylogenetic trees of each group provides a plausible scenario for the emergence of trHbs, by which the Group II trHb gene was the original gene, and the Group I trHb and Group III trHb genes were obtained via duplication and transfer events.

Amino Acid Sequence↗

PubMed2005

Protein dynamics in living cells.

A protein's structure is most often used to explain its function, but function also depends on dynamics. To date, protein dynamics have been studied only in vitro under dilute solution conditions where solute concentrations are typically less than 10 g/L, yet proteins function in a crowded environment where the solute concentration can exceed 400 g/L. Does the intracellular environment affect protein dynamics? The answer will help in assessing the biological significance of the NMR-derived dynamics data collected to date. We investigated fast protein dynamics inside living Escherichia coli by using in-cell NMR. The backbone dynamics of apocytochrome b5 were quantified using {1H}-15N nuclear Overhauser effect (nOe) measurements, which characterize motions on the pico- to nanosecond time scale. The overall trend of backbone dynamics remains the same in cells. Some of the nOe values differ, but most of the differences track the increased intracellular viscosity rather than a change in dynamics. Therefore, it appears that dilute solution steady-state {1H}-15N nOe measurements provide biologically relevant information about pico- to nanosecond backbone motion in proteins.

Escherichia coli↗

PubMed2005

Structural and dynamic properties of Synechocystis sp. PCC 6803 Hb revealed by reconstitution with Zn-protoporphyrin IX.

In its resting state, the truncated globin of the cyanobacterium Synechocystis sp. PCC 6803 exhibits hexacoordination of the heme iron, with His46 (E10) and His70 (F8, proximal) serving as axial ligands. Diatomic ligands displace the distal His46 (E10) from the ferric and ferrous iron and promote considerable structural changes in the B helix, E helix, and EF regions. Here, Zn(II)-substituted hemoglobin was used to explore the role of distal ligands in stabilizing the heme pocket structure. NMR data showed that the Zn ion was coordinated by the four pyrrole nitrogens and by His70 (F8) only. The proximal side of the Zn-porphyrin adopted a geometry recognizable as that of the wild-type protein. Decoordination of His46 (E10) to form the pentacoordinate Zn resulted in an incomplete transition to the conformation observed in the ferric, cyanide-bound protein. The NMR data also demonstrated that the H helix underwent complex dynamic processes near His117, a residue readily reacting with the wild-type heme 2-vinyl group in a post-translational modification.

Crystallography, X-Ray↗

PubMed2005

Structural divergence and distant relationships in proteins: evolution of the globins.

The globin family has long been known from studies of approximately 150-residue proteins such as vertebrate myoglobins and haemoglobins. Recently, this family has been enriched by the investigation of the sequences and structures of truncated globins, which have the same basic topology but are approximately 30 residues shorter and exhibit functions other than the familiar one of binding diatomic ligands. The divergence of protein sequences, structures and functions reveals Nature's exploration of the potential inherent in a folding pattern, that is, the topology of the native structure. The observation of what remains constant and what varies during the evolution of a protein family reveals essential features of structure and function. Study of proteins with a wide range of divergence can therefore sharpen our understanding of how different amino acid sequences can determine similar three-dimensional structures. Globins have provided, and continue to provide, interesting material for such studies.

Amino Acid Sequence↗

PubMed2005

Linkage between proton binding and folding in RNA: a thermodynamic framework and its experimental application for investigating pKa shifting.

Perturbation of pKa values can change the favored protonation states of the nucleobases at biological pH and thereby modulate the function of RNA and DNA molecules. In an effort to understand the driving forces for pKa shifting specific to nucleic acids, we developed a thermodynamic framework that relates proton binding to the nucleobases and the helix-coil transition. Key features that emerge from the treatment are a comprehensive description of all the actions of proton binding on RNA folding: acid and alkaline denaturation of the helix and pKa shifting in the folded state. Practical experimental approaches for measuring pKas from thermal denaturation experiments are developed. Microscopic pka values (where ka is the acid dissociation constant) for the unfolded state were determined directly by experiments on unstructured oligonucleotides, which led to a macroscopic pKa for the ensemble of unfolded states shifted toward neutrality. The formalism was then applied to pH-dependent UV melting data for model DNA oligonucleotides. Folded-state pka) values were in good agreement with the outcome of pH titrations, and the acid and alkaline denaturation regions were well described. The formalism developed here is similar to that of Draper and coworkers for Mg2+ binding to RNA, except that the unfolded state is described explicitly owing to the presence of specific proton-binding sites on the bases. A principal conclusion is that it should be possible to attain large pKa shifts by designing RNA molecules that fold cooperatively.

Binding Sites↗

PubMed2004

Cyanide binding to hexacoordinate cyanobacterial hemoglobins: hydrogen-bonding network and heme pocket rearrangement in ferric H117A Synechocystis hemoglobin.

The truncated hemoglobin (Hb) from the cyanobacterium Synechocystis sp. PCC 6803 is a bis-histidyl hexacoordinate complex in the absence of exogenous ligands. This protein can form a covalent cross-link between His117 in the H-helix and the heme 2-vinyl group. Cross-linking, the physiological importance of which has not been established, is avoided with the His117Ala substitution. In the present work, H117A Hb was used to explore exogenous ligand binding to the heme group. NMR and thermal denaturation data showed that the replacement was of little consequence to the structural and thermodynamic properties of ferric Synechocystis Hb. It did, however, decelerate the association of cyanide ions with the heme iron. Full complexation required hours, instead of minutes, of incubation at optical and NMR concentrations. At neutral pH and in the presence of excess cyanide, binding occurred with a first-order dependence on cyanide concentration, eliminating distal histidine decoordination as the rate-limiting step. The cyanide complex of the H117A variant was characterized for the conformational changes occurring as the histidine on the distal side, His46 (E10), was displaced. Extensive rearrangement allowed Tyr22 (B10) to insert in the heme pocket and Gln43 (E7) and Gln47 (E11) to come in contact with it. H-bond formation to the bound cyanide was identified in solution with the use of (1)H(2)O/(2)H(2)O mixtures. Cyanide binding also resulted in a change in the ratio of heme orientational isomers, in a likely manifestation of heme environment reshaping. Similar observations were made with the related Synechococcus sp. PCC 7002 H117A Hb, except that cyanide binding was rapid in this protein. In both cases, the (15)N chemical shift of bound cyanide was reminiscent of that in peroxidases and the orientation of the proximal histidine was as in other truncated Hbs. The ensemble of the data provided insight into the structural cooperativity of the heme pocket scaffold and pointed to the reactive 117 site of Synechocystis Hb as a potential determinant of biophysical and, perhaps, functional properties.

Alanine↗

PubMed2004

Insertion of the cytochrome b5 heme-binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture.

Under native conditions, apocytochrome b(5) exhibits a stable core and a disordered heme-binding region that refolds upon association with the cofactor. The termini of this flexible region are in close proximity, suggesting that loop closure may contribute to the thermodynamic properties of the apocytochrome. A chimeric protein containing 43 residues encompassing the cytochrome loop was constructed using the cyanobacterial photosystem I accessory protein E (PsaE) from Synechococcus sp. PCC 7002 as a structured scaffold. PsaE has the topology of an SH3 domain, and the insertion was engineered to replace its 14-residue CD loop. NMR and optical spectroscopies showed that the hybrid protein (named EbE1) was folded under native conditions and that it retained the characteristics of an SH3 domain. NMR spectroscopy revealed that structural and dynamic differences were confined near the site of loop insertion. Variable-temperature 1D NMR spectra of EbE1 confirmed the presence of a kinetic unfolding barrier. Thermal and chemical denaturations of PsaE and EbE1 demonstrated cooperative, two-state transitions; the stability of the PsaE scaffold was found only moderately compromised by the insertion, with a DeltaT(m) of 8.3 degrees C, a DeltaC(m) of 1.5 M urea, and a DeltaDeltaG degrees of 4.2 kJ/mole. The data implied that the penalty for constraining the ends of the inserted region was lower than the approximately 6.4 kJ/mole calculated for a self-avoiding chain. Extrapolation of these results to cytochrome b(5) suggested that the intrinsic stability of the folded portion of the apoprotein reflected only a small detrimental contribution from the large heme-binding domain.

Binding Sites↗

PubMed2004

A relationship between heme binding and protein stability in cytochrome b5.

Conformational changes and long-range effects are often observed in proteins when they associate with their ligands. In many cases, these structural perturbations are essential to function, and they are the result of complex networks of interactions. Here we used cytochrome b(5), a protein that undergoes extensive structural rearrangement upon heme binding, to seek a relationship between affinity for the cofactor and extent of refolding induced by its binding. Three variants of the water-soluble domain of the rat microsomal protein were chosen to affect the stability of the apoprotein or the holoprotein. Sequence alterations were introduced in the heme binding loop (type I mutations, D60R and (55)TENFED --> (55)TEPFEED, or PE), which is largely unstructured in the apoprotein state, and in the folded core of the apoprotein (type II mutation, P81A). Thermal and chemical denaturation experiments and heme transfer experiments were performed on these proteins. Type I mutations left the thermodynamic stability of the apoprotein unchanged. The first mutation (D60R) stabilized the holoprotein in a probable manifestation of enhanced helical propensity or improved electrostatic interactions. The second mutation (PE) decreased heme affinity and holoprotein stability in concert. For this protein, heme transfer experiments could be used to estimate the rate constant of heme loss from each of the heme orientational isomers. In contrast, the type II mutation resulted in a marked destabilization of the apoprotein but an intermediate effect on the holoprotein stability and heme affinity. These data supported that heme affinity could be modulated by the apoprotein stability and by specific residues remote from the heme binding site.

Animals↗

PubMed2004

Characterization of the heme-histidine cross-link in cyanobacterial hemoglobins from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002.

The recombinant product of the hemoglobin gene of the cyanobacterium Synechocystis sp. PCC 6803 forms spontaneously a covalent bond linking one of the heme vinyl groups to a histidine located in the C-terminal helix (His117, or H16). The present report describes the (1)H, (15)N, and (13)C NMR spectroscopy experiments demonstrating that the recombinant hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002, a protein sharing 59% identity with Synechocystis hemoglobin, undergoes the same facile heme adduct formation. The observation that the extraordinary linkage is not unique to Synechocystis hemoglobin suggests that it constitutes a noteworthy feature of hemoglobin in non-N(2)-fixing cyanobacteria, along with the previously documented bis-histidine coordination of the heme iron. A qualitative analysis of the hyperfine chemical shifts of the ferric proteins indicated that the cross-link had modest repercussions on axial histidine ligation and heme electronic structure. In Synechocystis hemoglobin, the unreacted His117 imidazole had a normal p K(a) whereas the protonation of the modified residue took place at lower pH. Optical experiments revealed that the cross-link stabilized the protein with respect to thermal and acid denaturation. Replacement of His117 with an alanine yielded a species inert to adduct formation, but inspection of the heme chemical shifts and ligand binding properties of the variant identified position 117 as important in seating the cofactor in its site and modifying the dynamic properties of the protein. A role for bis-histidine coordination and covalent adduct formation in heme retention is proposed.

Acids↗

PubMed2004

Structural properties of cyanobacterial hemoglobins: the unusual heme-protein cross-link of Synechocystis sp. PCC 6803 Hb and Synechococcus sp. PCC 7002 Hb.

The truncated hemoglobins from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 ligate the heme iron with two axial histidines (HisF8 and HisE10). In addition, these two proteins are able to form a heme-protein cross-link between a vinyl substituent and a histidine at position 16 of the H helix. The product is a protein with improved resistance to thermal and acid denaturation.

Bacterial Proteins↗

PubMed2003

A de novo redesign of the WW domain.

We have used a sequence prediction algorithm and a novel sampling method to design protein sequences for the WW domain, a small beta-sheet motif. The procedure, referred to as SPANS, designs sequences to be compatible with an ensemble of closely related polypeptide backbones, mimicking the inherent flexibility of proteins. Two designed sequences (termed SPANS-WW1 and SPANS-WW2), using only naturally occurring L-amino acids, were selected for study and the corresponding polypeptides were prepared in Escherichia coli. Circular dichroism data suggested that both purified polypeptides adopted secondary structure features related to those of the target without the aid of disulfide bridges or bound cofactors. The structure exhibited by SPANS-WW2 melted cooperatively by raising the temperature of the solution. Further analysis of this polypeptide by proton nuclear magnetic resonance spectroscopy demonstrated that at 5 degrees C, it folds into a structure closely resembling a natural WW domain. This achievement constitutes one of a small number of successful de novo protein designs through fully automated computational methods and highlights the feasibility of including backbone flexibility in the design strategy.

Algorithms↗

PubMed2002

The solution structure of the recombinant hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803 in its hemichrome state.

The product of the cyanobacterium Synechocystis sp. PCC 6803 gene slr2097 is a 123 amino acid polypeptide chain belonging to the truncated hemoglobin family. Recombinant, ferric heme-reconstituted Synechocystis sp. PCC 6803 hemoglobin displays bis-histidine coordination of the iron ion. In addition, this protein is capable of covalently attaching a reactive histidine to the heme 2-vinyl group. The structure of the protein in the low-spin ferric state with intact vinyl substituents was solved by NMR methods. It was found that the structure differs from that of known truncated hemoglobins primarily in the orientation of the E helix, which carries His46 (E10) as the distal ligand to the iron; the length and orientation of the F helix, which carries His70 (F8) as the proximal ligand to the iron; and the H-helix, which carries His117 (H16), the reactive histidine. Regions of enhanced flexibility include the short A helix, the loop connecting the E and F helices, and the last seven residues at the carboxy end. The structural data allowed for the rationalization of physical properties of the cyanobacterial protein, such as fast on-rate for small ligand binding, unstable apoprotein fold, and cross-linking ability. Comparison to the truncated hemoglobin from the green alga Chlamydomonas eugametos also suggested how the endogenous hexacoordination affected the structure.

Amino Acid Sequence↗

PubMed2002

Novel histidine-heme covalent linkage in a hemoglobin.

When treated with dithionite at neutral pH, the recombinant hemoglobin from Synechocystis sp. PCC 6803 reconstituted with ferric heme undergoes a rapid chemical reaction resulting in the attachment of the heme group to the polypeptide chain. The nature of the cross-linked species was studied by NMR and mass spectral methods. 1H NMR data indicated that the 2-vinyl group was the reacting moiety of the heme. Mass spectrometry of pepsin digests located the site of attachment within a 12-mer at the C-terminal end of the protein. Homonuclear and 1H-15N NMR data identified the modified residue as His117, which underwent addition to the vinyl Calpha through the imidazole Nepsilon. Dithionite treatment of the globin reconstituted with Zn protoporphyrin IX sample did not lead to 2-vinyl group modification, suggesting that the chemical reduction of the heme iron facilitated the attachment.

Apoproteins↗

PubMed2002

Truncated hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002: evidence for hexacoordination and covalent adduct formation in the ferric recombinant protein.

The glbN gene for the hemoglobin of Synechoccocus sp. PCC 7002, a cyanobacterium incapable of nitrogen fixation, was cloned and overexpressed in Escherichia coli. The 123-residue protein was purified from inclusion bodies and reconstituted with iron protoporphyrin IX to obtain the ferric form of the holoprotein. Mass spectrometric analysis confirmed the identity of the polypeptide. NMR and optical data demonstrated that the protein so prepared contained a hexacoordinate heme group, as observed in the related globin from Synechocystis sp. PCC 6803 [Scott, N. L., and Lecomte, J. T. J. (2000) Protein Sci. 9, 587-597]. The data were consistent with a similar bis-histidine coordination scheme involving His46 (E10) on the distal side and His70 (F8) on the proximal side. Several aromatic residues were identified in the vicinity of the heme and were used to establish the orientation of the prosthetic group in the polypeptide matrix. In this protein, as in that from Synechocystis sp. PCC 6803, there was a marked preference for the heme orientation in which pyrroles C and D contact the C-E corner of the protein. Both hemoglobins were found capable of forming a product in which the heme is cross-linked to the polypeptide through modification of a vinyl group.

Amino Acid Sequence↗