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D L Turner

Publications and source records attributed to D L Turner.

At least 73 records · Page 4Linked to original sources
PubMed1994

Homotropic and heterotropic cooperativity in the tetrahaem cytochrome c3 from Desulfovibrio vulgaris.

The thermodynamic parameters which govern the homotropic (e-/e-) and heterotropic (e-/H+) cooperativity in the tetrahaem cytochrome c3 isolated from Desulfovibrio vulgaris (Hildenborough) were determined, using the paramagnetic shifts of haem methyl groups in the NMR spectra of intermediate oxidized states at different pH levels. A model is put forward to explain how the network of positive and negative cooperativities between the four haems and acid/base group(s) enables the protein to achieve a proton-assisted 2e- step.

Cytochrome c Group↗
PubMed1994

An unusual conformation of the methionine haem ligand in cytochrome cL established by two-dimensional 1H-NMR.

A complete relaxation-matrix analysis of NOESY cross-peak intensities was used to determine the conformation of the methionine ligand to the haem group in two ferrocytochromes cL from Methylophilus methylotrophus and Methylobacterium extorquens, including the configuration at the sulphur. The conformation of the axial methionine is of a type reported only for the cytochromes c5 from Pseudomonas mendocina and Azotobacter vinelandii. Although the conformation of the methionine is unusual, the paramagnetic shifts of the haem methyl proton resonances in the oxidized proteins indicate that the electronic structure of the haem groups is similar to that found in the mitochondrial type of cytochrome c.

Computer Simulation↗
PubMed1994

Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate.

In Drosophila, the proneural genes of the achaete-scute complex encode transcriptional activators that can commit cells to a neural fate. We have isolated cDNAs for two Xenopus achaete-scute homologs, ASH3a and ASH3b, which are expressed in a subset of central nervous system (CNS) neuroblasts during early neurogenesis. After expressing either ASH3 protein in developing Xenopus embryos, we find enlargement of the CNS at the expense of adjacent non-neural ectoderm. Analysis of molecular markers for neural, epidermal, and neural crest cells indicates that CNS expansion occurs as early as neural plate formation. ASH3-dependent CNS enlargement appears to require neural induction, as it does not occur in animal cap explants. Inhibition of DNA synthesis shows that additional CNS tissue does not depend on cell division--rather it reflects conversion of prospective neural crest and epidermal cells to a neural fate. The differentiation of the early forming primary neurons also seems to be prevented by ASH3 expression. This may be secondary to the observed activation of Xotch transcription by ASH3.

Amino Acid Sequence↗
PubMed1993

Characterization of the structure and redox behaviour of cytochrome c3 from Desulfovibrio baculatus by 1H-nuclear-magnetic-resonance spectroscopy.

Complete assignment of the aromatic and haem proton resonances in the cytochromes c3 isolated from Desulfovibrio baculatus strains (Norway 4, DSM 1741) and (DSM 1743) was achieved using one- and two-dimensional 1H n.m.r. Nuclear Overhauser enhancements observed between haem and aromatic resonances and between resonances due to different haems, together with the ring-current contributions to the chemical shifts of haem resonances, support the argument that the haem core architecture is conserved in the various cytochromes c3, and that the X-ray structure of the D. baculatus cytochrome c3 is erroneous. The relative orientation of the haems for both cytochromes was determined directly from n.m.r. data. The n.m.r. structures have a resolution of approximately 0.25 nm and are found to be in close agreement with the X-ray structure from D. vulgaris cytochrome c3. The proton assignments were used to relate the highest potential to a specific haem in the three-dimensional structure by monitoring the chemical-shift variation of several haem resonances throughout redox titrations followed by 1H n.m.r. The haem with highest redox potential is not the same as that in other cytochromes c3.

Bacterial Proteins↗
PubMed1993

Structural studies on Desulfovibrio gigas cytochrome c3 by two-dimensional 1H-nuclear-magnetic-resonance spectroscopy.

Several aromatic amino acid residues and haem resonances in the fully reduced form of Desulfovibrio gigas cytochrome c3 are assigned, using two-dimensional 1H n.m.r., on the basis of the interactions between the protons of the aromatic amino acids and the haem protons as well as the intrahaem distances known from the X-ray structure [Kissinger (1989) Ph.D. Thesis, Washington State University]. The interhaem interactions observed in the n.m.r. spectra are in full agreement with the D. gigas X-ray structure and also with the n.m.r. data from Desulfovibrio vulgaris (Hildenborough) [Turner, Salgueiro, LeGall and Xavier (1992) Eur. J. Biochem. 210, 931-936]. The good correlation between the calculated ring-current shifts and the observed chemical shifts strongly supports the present assignments. Observation of the two-dimensional nuclear-Overhauser-enhancement spectra of the protein in the reduced, intermediate and fully oxidized stages led to the ordering of the haems in terms of their midpoint redox potentials and their identification in the X-ray structure. The first haem to oxidize is haem I, followed by haems II, III and IV, numbered according to the Cys ligand positions in the amino acid sequences [Mathews (1985) Prog. Biophys. Mol. Biol. 54, 1-56]. Although the haem core architecture is the same for the different Desulfovibrio cytochromes c3, the order of redox potentials is different.

Bacterial Proteins↗
PubMed1993

Characterization of the haem environment in Methylophilus methylotrophus ferricytochrome c" by 1H-NMR.

Two-dimensional NMR techniques have been used to assign proton resonances in the haem cavity of Methylophilus methylotrophus cytochrome c", a monohaem protein with bis-histidinyl ligation which has been shown to couple electron and proton transfer. All the assignments were made directly for the oxidized paramagnetic form of the cytochrome. Nearly all of the haem protons (90%) and the protons of both axial ligands have been assigned; the side-chain protons from four other residues in the haem pocket have also been identified. The data indicate a highly symmetric unpaired-electron distribution in the haem group, which agrees with a perpendicular orientation of the axial imidazole planes. The two haem propionate groups have contrasting degrees of exposure to the solvent, with the propionate group at position 13 being highly exposed. To obtain information on the dynamics of the haem environment, measurements of the 1H/2H-exchange rates of amide protons located in the haem cavity were performed. The two faces of the haem are found to differ markedly with respect to water accessibility. All of this information, together with additional protein sequencing data, indicates that His52 remains attached upon reduction and that the redox-linked protonation occurs via a channel running through the haem cleft on the opposite face.

Amino Acid Sequence↗
PubMed1993

Pitfalls in assigning heme axial coordination by EPR. c-Type cytochromes with atypical Met-His ligation.

Different monohemic c-type cytochromes were analyzed by visible, EPR and 1H NMR spectroscopies. While the visible and NMR data show unambiguously that the heme iron has a Met-His heme axial coordination, the EPR data indicate an axial ligand field typical of that for a bis-histidinyl ligation. The validity of the widely used EPR methods for the determination of the heme iron axial coordination, based on the crystal field parameters (tetragonality and rhombicity), is questioned.

Bacterial Proteins↗
PubMed1993

1H- and 13C-NMR investigation of redox-state-dependent and temperature-dependent conformation changes in horse cytochrome c.

The redox-state dependent changes in chemical shift, which have been measured for almost 100 CHn groups in the 13C-NMR spectra of horse cytochrome c [Santos, H., and Turner, D. L. (1992) Eur. J. Biochem. 206, 721-728], have been used to investigate the nature of the redox-related change in conformation. Apart from the haem and its axial ligands, the shifts are found to be dominated by the electron-nuclear dipolar coupling in the oxidised form, as was the case in 1H-NMR studies. These pseudocontact shifts are well described by using an empirically determined magnetic susceptibility tensor in conjunction with atomic coordinates for the horse cytochrome c. The groups which fit least well are located in the vicinity of Trp59. Comparison between 1H and 13C shifts and their temperature dependence shows that the differences from expectation based on a single structure for both oxidation states are caused largely by changes in the diamagnetic contribution to the chemical shifts. Since these are different for 1H and 13C resonances they indicate, independently from crystal structure data, some redox-related movement of the protein under the haem. The significance of these results for understanding electron transfer pathways is discussed. Finally, the temperature dependence of the pseudocontact shifts in the range 30-50 degrees C is shown to be anomalous. Approximately half of the anomalous effect may be attributed to Zeeman mixing of the electronic wavefunctions with a spin-orbit coupling constant lambda = 241 cm-1, while the other half is attributed to thermal expansion of the protein.

Animals↗
PubMed1993

Evaluation of 13C and 1H Fermi contact shifts in horse cytochrome c. The origin of the anti-Curie effect.

Many ferricytochromes c exhibit a peculiar effect in which the 1H chemical shifts of the haem methyl groups appear in pairs and, although the paramagnetic shifts of the two groups with the larger shifts decrease with temperature, those of the pair with the smaller shifts actually increase. Recent NMR studies [Santos, H. and Turner, D. L. (1992) Eur. J. Biochem. 206, 721-728] gave 1H and 13C assignments for most of the haem substituents and the axial ligands in horse cytochrome c at 30 degrees C and 50 degrees C in both oxidation states. These data are used together with an empirically determined magnetic susceptibility tensor to evaluate the Fermi contact contribution to the paramagnetic shift and hence map the delocalization of the unpaired electron. The anti-Curie effect is explained by a Boltzmann distribution between partially filled porphyrin 3e(pi) molecular orbitals with an energy difference of 3 kJ/mol. The fact that the energy gap is small with respect to the energy of binding to the electron transfer partners calls into question the significance of the asymmetry of the electron distribution in the electron transfer process.

Animals↗
PubMed1993

Limitations to VO2max in humans after blood retransfusion.

Seven young, healthy male subjects performed maximal exercise on a cycloergometer with central venous and arterial catheters, before and after autologous retransfusion of red blood cells. Maximal oxygen consumption (VO2max), blood gas composition and haemodynamic variables were measured, in order to test the hypothesis of monofactorial vs. polyfactorial VO2max limitation. Autologous blood retransfusion led to significant increases in haemoglobin concentration and consequently arterial oxygen concentration during maximal exercise, while maximal cardiac output, heart rate and stroke volume were not significantly changed. The relationship between maximal oxygen delivery (cardiac output.arterial oxygen concentration; (Q.CaO2)max and maximal oxygen consumption in this study was VO2max (L.min-1) = 0.02 + 0.64.(Q.CaO2)max (L.min-1), the slope being significantly less than unity. These results suggest that (Q.CaO2)max plays but a fractional role in limiting VO2max, in agreement with recent models concerning the resistance to oxygen flow in the respiratory system (di Prampero and Ferretti, Respir. Physiol. 80: 113-128, 1990). The relative increase in VO2max after blood retransfusion matched the relative increase in 'aerobic performance', measured as the maximal power output that could be maintained aerobically for 30 min. Furthermore, the increase in maximal power output (15 +/- 3 watts) could account for almost all of the extra oxygen consumption. This match suggests that there is an inability to fully utilize muscle oxidative capacity in the normocythaemic state.

Adult↗
PubMed1992

Structural studies of Desulfovibrio vulgaris ferrocytochrome c3 by two-dimensional NMR.

Two-dimensional NMR has been used to make specific assignments for the four haems in Desulfovibrio vulgaris (Hildenborough) ferrocytochrome c3 and to determine their haem core architecture. The NMR signals from the haem protons were assigned according to type using two-dimensional NMR experiments which led to four sets of signals, one for each of the haems. Specific assignments were obtained by calculating the ring current shifts which arise from other haems and aromatic residues. Observation of interhaem NOEs confirmed the assignments and established that the relative orientation of the haems is identical to that found in the crystal structure of D. vulgaris (Miyazaki F.) ferricytochrome c3. Assignments were also made for all the aromatic residues except for the haem ligands and F20, which is shifted under the main envelope of signals. The NOEs observed between these aromatic protons and haem protons confirm the similarity between the structures in solution and in the crystal. The assignments reported here are the basis for the cross-assignments of the four microscopic haem redox potentials to specific haems in the protein structure [Salgueiro, C. A., Turner, D. L., Santos, H., LeGall, J. and Xavier, A. V. (1992) FEBS Lett., in the press]

Amino Acid Sequence↗
PubMed1992

Assignment of the redox potentials to the four haems in Desulfovibrio vulgaris cytochrome c3 by 2D-NMR.

Using 2D-NMR the four haems of Desulfovibrio vulgaris (Hildenborough) cytochrome c3 within the X-ray structure were fully cross assigned according to their redox potential. The strategy used was based on a complete network of chemical exchange connectivities between the NMR signals obtained for all oxidation levels to the corresponding ones in the fully reduced spectrum [1992, Eur. J. Biochem., in press]. This unequivocal cross-assignment disagrees with earlier results obtained for the similar protein from Desulfovibrio vulgaris (Miyazaki F.).

Cytochrome c Group↗
PubMed1992

Revision of the haem-core architecture in the tetraheam cytochrome c3 from Desulfovibrio baculatus by two-dimensional 1H NMR.

The haem-core architecture in cytochrome c3 isolated from Desulfovibrio baculatus (Norway 4) was probed using two-dimensional 1H NMR. Interhaem connectivities detected in NOE spectroscopy experiments performed at short mixing times are incompatible with the structure of the protein determined by X-ray crystallography, but agree instead with the haem arrangement found in cytochrome c3 from Desulfovibrio vulgaris (Miyazaki). These experiments show unequivocally that the relative orientation of the four haems in the two proteins is the same and does not involve the 180 degrees rotation of haems I and IV indicated in the X-ray structure determined for the cytochrome c3 from D. baculatus (Norway 4).

Cytochrome c Group↗
PubMed1992

Involvement of a labile axial histidine in coupling electron and proton transfer in Methylophilus methylotrophus cytochrome c''.

Methylophilus methylotrophus cytochrome c'' is an unusual monohaem protein (15 kDa) undergoing a redox-linked spin-state transition [Santos, H. & Turner, D. L. (1988) Biochim. Biophys. Acta 954, 277-286]. The midpoint redox potential of cytochrome c" was measured over the pH range 4-10. The pH dependence of the midpoint redox potential was interpreted in terms of a model that considers the redox-state dependence of the ionization of two distinct and non-interacting protonated groups in the protein. This analysis led to the following pKa values within the pH range studied: pKa10 = 6.4, pKa1r = 5.4 and pKa2r = 8.1. Proton-NMR spectroscopy was used to assist the characterization of the two ionizing groups responsible for the observed redox-Bohr effect: the group ionizing with a lower pKar was assigned to a haem propionic acid substituent and the other to the axial histidine ligand which becomes detached upon reduction, which has a pKa0 too low to be measured. It is shown that M. methylotrophus cytochrome c" is able to couple electron and proton transfer in the physiological pH range through a mechanism involving reversible change in the haem-iron coordination. Possible implications for the physiological role of the protein are discussed.

Amino Acids↗
PubMed1992

Structure determination of a novel cyclic phosphocompound isolated from Desulfovibrio desulfuricans.

The structure of a novel diphosphodiester compound recently detected in Desulfovibrio desulfuricans cells [Santos, Fareleira, Pedregal, LeGall & Xavier (1991) Eur. J. Biochem. 201, 283-287] was fully elucidated using a combination of n.m.r. techniques in aqueous and in methanolic solutions. The novel metabolite was identified as 3-methyl-1,2,3,4-tetrahydroxybutane-1,3-cyclic bisphosphate, and the minimum energy conformation is presented. The two chiral centres have the relative configuration RS.

Butanols↗
PubMed1992

13C and proton NMR studies of horse cytochrome c. Systematic assignment of methyl and methine resonances in both oxidation states.

The CHn groups in the aliphatic side chains of horse cytochrome c have been characterized according to the chemical shifts of both 13C-NMR and 1H-NMR signals, their temperature dependence and the number of attached protons, n. The primary assignments of resonances from the 55 side-chain methyl and the 27 methine groups were obtained directly for the oxidised and the reduced forms. Specific assignments of the 13C resonances were obtained through shift-correlation experiments and comparison with earlier 1H-NMR studies, by further measurements of proton-proton interactions, or by elimination. Comparison of the paramagnetic shifts of carbon and protons indicates a small redox-related change of conformation in the vicinity of Trp59 and a significant expansion of the protein over 30-50 degrees C.

Amino Acids↗
PubMed1992

Site-directed mutagenesis of the C-terminal portion of reovirus protein sigma 1: evidence for a conformation-dependent receptor binding domain.

Oligonucleotide site-directed mutagenesis was used to modify the type 3 (T3) reovirus cell attachment protein sigma 1 at residues located in the three regions (designated C, D, and E in the C-terminal one-third of sigma 1) that are highly conserved between the three reovirus serotypes. Of the eight residues targeted for mutagenesis, five (one in region C, and two each in regions D, and E) are conserved among all three proteins. Wild-type (wt) and mutant sigma 1 forms were synthesized in an vitro transcription/translation system and subjected to structural and functional analysis. None of the mutations affected the ability of sigma 1 to form trimers. However, mutation (all representing drastic changes) in any of the five triply conserved residues (Tyr326, Asn369, Phe370, Tyr450, and Pro451) caused a complete or partial abrogation of sigma 1 cell binding function, whereas mutation in any of the other three residues (Ser325, Ser327, and Asp365) had no adverse effect. The structural integrity of the mutant proteins was then probed using trypsin, chymotrypsin, and a neutralizing monoclonal anti-sigma 1 antibody. In all cases, the loss of cell binding function was associated with a drastic conformational change in the C-terminal globular head of sigma 1. These results suggest that conserved residues in the three highly conserved regions in the C-terminal portion of sigma 1 play important structural and functional roles and are involved in proper head folding and generation of a conformation-dependent receptor binding domain.

Amino Acid Sequence↗