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P Gans

Publications and source records attributed to P Gans.

At least 19 recordsLinked to original sources
PubMed2001

Magnetic susceptibility tensor and heme contact shifts determinations in the Rhodobacter capsulatus ferricytochrome c': NMR and magnetic susceptibility studies.

The 1H and 15N resonances of the carbon monoxide complex of ferrocytochrome c' of Rhodobacter capsulatus, a ferrous diamagnetic heme protein, have been extensively assigned by TOCSY-HSQC, NOESY-HSQC, and HSQC-NOESY-HSQC 3D heteronuclear experiments performed on a 7 mM sample labeled with 15N. Based on short-range and medium-range NOEs and H(N) exchange rates, the secondary structure consists of four helices: helix 1 (3-29), helix 2 (33-48), helix 3 (78-101), and helix 4 (103-125). The 15N, 1HN, and 1H(alpha) chemical shifts of the CO complex form are compared to those of the previously assigned oxidized (or ferric) state. From the chemical shift differences between these redox states, the orientation and the anisotropy of the paramagnetic susceptibility tensor have been determined using the crystallographic coordinates of the ferric state. The chi-tensor is axial, and the orientation of the z-axis is approximately perpendicular to the heme plane. The paramagnetic chemical shifts of the protons of the heme ligand have been determined and decomposed into the Fermi shift and dipolar shift contributions. Magnetic susceptibility studies in frozen solutions have been performed. Fits of the susceptibility data using the model of Maltempo (Maltempo, M. M. J. Chem. Phys. 1974, 61, 2540-2547) are consistent with a rather low contribution of the S = 3/2 spin state over the range of temperatures and confirm the value of the axial anisotropy. Values in the range 10.4-12.5 cm(-1) have been inferred for the axial zero-field splitting parameter (D). Analysis of the contact shift and the susceptibility data suggests that cytochrome c' of Rb. capsulatus exhibits a predominant high-spin character of the iron in the oxidized state at room temperature.

Cytochrome c Group↗
PubMed2001

NMR and CD conformational studies of the C-terminal 16-peptides of Pseudomonas aeruginosa c551 and Hydrogenobacter thermophilus c552 cytochromes.

The 16-amino acid sequences of the C-terminal helices of the homologous bacterial cytochromes c551 from Pseudomonas aeruginosa and C552 from Hydrogenobacter thermophilus were synthesized and their solution structure studied. Circular dichroism and NMR experiments in aqueous solution have shown the presence of alpha-helices and 3(10)-helices. The populations of helical structures in phosphate buffer, pH 3.5, 293 K, were 21% for c551 and 20% for c552, but increased to 56.7 and 48%, respectively, in 50% aqueous 2,2,2-trifluoroethanol. An isodichroic point was observed at 203 nm in CD spectra for the helix/coil transition in mixtures of water/2,2,2-trifluoroethanol. NMR spectra in phosphate buffer show the presence of both alpha- and 3(10)-helical structures. In water/2,2,2-trifluoroethanol (50:50) alpha-helices are predominant. CD temperature-dependency studies indicate that both peptides exhibit the same cooperativity for the transition in water/2,2,2-trifluoroethanol (50:50). The experimental data show that the amino acid substitutions do not favor heat resistance of the secondary structure of the c552 C-terminal helix at the local level. Instead, they optimize nonlocal contacts of the polypeptide chain, which stabilize the tertiary structure in the native protein.

Algorithms↗
PubMed2000

Combined structural and biochemical analysis of the H-T complex in the glycine decarboxylase cycle: evidence for a destabilization mechanism of the H-protein.

The lipoate containing H-protein plays a pivotal role in the catalytic cycle of the glycine decarboxylase complex (GDC), undergoing reducing methylamination, methylene transfer, and oxidation. The transfer of the CH(2) group is catalyzed by the T-protein, which forms a 1:1 complex with the methylamine-loaded H-protein (Hmet). The methylamine group is then deaminated and transferred to the tetrahydrofolate-polyglutamate (H(4)FGlu(n)) cofactor of T-protein, forming methylenetetrahydrofolate-polyglutamate. The methylamine group is buried inside the protein structure and highly stable. Experimental data show that the H(4)FGlu(n) alone does not induce transfer of the methylene group, and molecular modeling also indicates that the reaction cannot take place without significant structural perturbations of the H-protein. We have, therefore, investigated the effect of the presence of the T-protein on the stability of Hmet. Addition of T-protein without H(4)FGlu(n) greatly increases the rate of the unloading reaction of Hmet, reducing the activation energy by about 20 kcal mol(-1). Differences of the (1)H and (15)N chemical shifts of the H-protein in its isolated form and in the complex with the T-protein show that the interaction surface for the H-protein is localized on one side of the cleft where the lipoate arm is positioned. This suggests that the role of the T-protein is not only to locate the tetrahydrofolate cofactor in a position favorable for a nucleophilic attack on the methylene carbon but also to destabilize the H-protein in order to facilitate the unlocking of the arm and initiate the reaction.

Amino Acid Oxidoreductases↗
PubMed1999

A coil-helix instead of a helix-coil motif can be induced in a chloroplast transit peptide from Chlamydomonas reinhardtii.

A synthetic peptide MQVTMKSSAVSGQRVGGARVATRSVRRAQLQV corresponding to the 32 amino acid chloroplast transit sequence of the ribulose bisphosphatase carboxylase/oxygenase activase preprotein from Chlamydomonas reinhardtii, required for translocation through the envelope of the chloroplast, has been characterized structurally using CD and NMR under the same experimental conditions as used previously for the 32 amino acid presequence of preferredoxin from the same organism [Lancelin, J.-M., Bally, I., Arlaud, G. J., Blackledge, M., Gans, P., Stein, M. & Jacquot, J.-P. (1994) FEBS Lett. 343, 261-266]. The peptide is found to undergo a conformational transition in aqueous 2,2,2-trifluoroethanol, characterized by three turns of amphiphilic alpha-helix in the C-terminal region preceded by a disordered coil in the N-terminal region. Compared with the preferredoxin transit peptide, the helical and coiled domains are arranged in the reverse order along the peptide sequence, but the positively charged groups are distributed analogously as well as the hydrophobic residues within the amphiphilic alpha-helix. It is proposed that such coil-helix or helix-coil motifs, occasionally repeated, could be an intrinsic structural feature of chloroplastic transit peptides, adapted to the proper translocase and possibly to each nuclear-encoded chloroplast preproteins. This feature may distinguish chloroplastic transit sequences from the other organelle-targeting peptides in the eukaryotic green alga C. reinhardtii, particularly the mitochondrial transit sequences.

Amino Acid Sequence↗
PubMed1999

Structural and functional characterization of H protein mutants of the glycine decarboxylase complex.

The mitochondrial glycine decarboxylase complex (GDC) consists of four component enzymes (P, H, T, and L proteins) involved in the breakdown of glycine. In order to investigate structural interactions involved in the stabilization of the methylamine-loaded H protein (a transient species in the GDC reaction), we designed several mutants of H apoprotein. Structural analysis of the wild-type and mutants of H apoprotein emphasized the necessity to carefully assess, by biophysical techniques, the correct folding of mutated proteins prior to investigate their biochemical properties. The correctly folded wild-type and mutants of H apoprotein were in vitro lipoylated and then characterized in the context of GDC reaction by studying the reconstituted complex and partial reactions. We showed that Val(62) and Ala(64), surrounding the lipoyl-lysine, play an important role in the molecular events that govern the reaction between P and H protein but do not intervene in the recognition of the binding site of lipoic acid by lipoyl ligase. The biochemical results obtained with the HE14A mutant of H protein pointed out the major role of the Glu(14) amino acid residue in the GDC catalysis and highlighted the importance of the ionic and hydrogen bounds in the hydrophobic cleft of H protein for the stabilization of the methylamine-loaded lipoyl arm.

Amino Acid Oxidoreductases↗
PubMed1999

Investigation of the local structure and dynamics of the H subunit of the mitochondrial glycine decarboxylase using heteronuclear NMR spectroscopy.

The lipoate-dependent H protein plays a pivotal role in the catalytic cycle of the glycine decarboxylase complex (GDC), undergoing reducing methylamination, methylene transfer, and oxidation. The local structure and backbone dynamics of the methylamine-loaded H (Hmet), oxidized H (Hox), and H apoprotein (Hapo) have been investigated in solution. Filtered NOESY experiments using a [13C]Hmet as well as comparison of the heteronuclear shifts between the Hox and Hmet proteins demonstrate that the methylamine group is located inside a cleft of the protein. Furthermore, this group appears to be locked in this configuration as indicated by the high value of the activation energy (37 kcal/mol) of the global unloading reaction and by its restricted mobility, deduced from 13C relaxation measurements. Comparisons of the 1H and 15N chemical shifts and 15N relaxation in the three forms suggest that part of the lipoyl-lysine arm interacts with the protein polypeptide in the Hox and Hmet. The major change induced by the loading of the methylamine group concerns the C-terminal helix whose mobility becomes completely restricted compared to those of the Hox and Hapo. This C-terminal helix exhibits different reorientational characteristics in the three forms, which can be explained in the Hapo by a model consisting of a twisting motion about an axis passing through the helix. Our results indicate that the model of a freely swinging arm proposed for other lipoate-containing proteins is not acceptable in solution for the GDC. The implication of this observation in terms of the mechanism of the interaction of the H protein with the T protein, its physiological partner during the catalytic cycle, is discussed.

Amino Acid Oxidoreductases↗
PubMed1998

NMR structures of the C-terminal end of human complement serine protease C1s.

Synthetic peptides derived from the C-terminal end of the human complement serine protease C1s were analysed by circular dichroism and nuclear magnetic resonance (NMR) spectroscopy. Circular dichroism indicates that peptides 656-673 and 653-673 are essentially unstructured in water and undergo a coil-to-helix transition in the presence of increasing concentrations of trifluoroethanol. Two-dimensional NMR analyses performed in water/trifluoroethanol solutions provide evidence for the occurrence of a regular alpha-helix extending from Trp659 to Ser668 (peptide 656-673), and from Tyr656 to Ser668 (peptide 653-673), the C-terminal segment of both peptides remaining unstructured under the conditions used. Based on these and other observations, we propose that the serine protease domain of C1s ends in a 13-residue alpha-helix (656Tyr-Ser668) followed by a five-residue C-terminal extension. The latter appears to be flexible and is probably locked within C1s through a salt bridge involving Glu672.

Amino Acid Sequence↗
PubMed1997

Solution structure of the cellulose-binding domain of the endoglucanase Z secreted by Erwinia chrysanthemi.

Two-dimensional proton nuclear magnetic resonance spectroscopy has been used to determine the three-dimensional structure of the 62 amino acid C-terminal cellulose-binding domain (CBD) of the endoglucanase Z (CBDEGZ), secreted by Erwinia chrysanthemi. An experimental data set comprising 958 interproton nOe-derived restraints was used to calculate 23 structures. The calculated structures have an average root-mean-square deviation between Cys4 and Cys61 of 0.91 +/- 0.11 A for backbone atoms and 1.18 +/- 0.12 A for the heavy atoms. The CBDEGZ exhibits a skiboot shape based mainly on a triple antiparallel beta-sheet perpendicular to a less-ordered summital loop. Three aromatic rings (Trp18, Trp43, and Tyr44) are localized on one face of the protein and are exposed to the solvent in a conformation compatible with a cellulose-binding site. Based on its original folding, we have been able to relate the CBD sequence to those of several domains of unknown function occurring in several bacterial chitinases as well as other proteins. This study also provides a structural basis for analyzing the secretion-related information specific to the CBDEGZ.

Amino Acid Sequence↗
PubMed1996

NMR structures of a mitochondrial transit peptide from the green alga Chlamydomonas reinhardtii.

The 26-amino-acid pre-sequence of the ATP synthase beta subunit that directs the protein from the cytosol to mitochondria in the unicellular green alga Chlamydomonas reinhardtii has been synthesised and analysed using NMR spectroscopy/circular dichroism and compared to a chloroplast transit peptide from the same organism. The results demonstrate that the peptide, though mainly unstructured in water, undergoes a strong conformational change in a 36% water/64% 2,2,2-trifluoroethanol mixture. In this solvent condition, an alpha-helix was characterised by NMR from residue 2 to 26. Structure calculations under NMR restraints lead to a population of models of which 60% are kinked at position 9-10. Structural analysis indicates two hydrophobic sectors on the models with a discontinuity at the 9-10 kink level. The structures suggest a different interaction mode with the mitochondrial membrane compared to the chloroplast transit peptide.

Amino Acid Sequence↗
PubMed1996

Sequential 1H and 15N NMR resonance assignment and secondary structure of ferrocytochrome c2 from Rhodobacter sphaeroides.

Sequence-specific 1H and 15N assignments have been made for the amino acids of the ferrocytochrome c2 from Rhodobacter sphaeroides. Initial assignments were made by analysis of a series of homonuclear 2D COSY, TOCSY, and NOESY spectra obtained with the unlabeled protein. 2D and 3D 1H-15N correlated spectra obtained for a uniformly 15N-labeled ferrocytochrome c2 were used to confirm and extend the assignments. Partial 13C assignments have also been made by means of HSQC experiments on 13C at natural abundance, in particular for about two-thirds of the 13C alpha. Medium-range NOE connectivities, together with 3J(HC alpha NH) coupling constants, indicated the presence of five helices at positions 6-16, 60-68, 74-82, 84-91, and 109-120. No other regular secondary structure was observed. This folding is similar to that previously observed for the ferrocytochrome c2 of Rhodobacter capsulatus in solution, which exhibits approximately 50% sequence identity. Moreover, the rotation rates of the aromatic rings of phenylalanine or tyrosine, when conserved, were similar to those observed for R. capsulatus. Furthermore, C alpha H chemical shifts, which are sensitive to the secondary structure and ring current effects of the heme, appear to be very similar for the two proteins. Consequently, the solution structure of R. sphaeroides ferrocytochrome c2 appears to be very similar to that of R. capsulatus ferrocytochrome c2. These results are compared with the X-ray crystal structure of the R. sphaeroides ferrocytochrome c2.

Amino Acid Sequence↗
PubMed1995

Nuclear magnetic resonance as a tool for determining protonation constants of natural polyprotic bases in solution.

The acid-base properties of the tetramine 1,5,10,14-tetraazatetradecane H2N(CH2)3NH(CH2)4NH(CH2)3NH2 (spermine) in deuterated water have been studied at 40 degrees C at various pD values by means of NMR spectroscopy. Both one-dimensional 13C[1H] spectra and two-dimensional 1H/13C heterocorrelation spectra with inverse detection have been recorded. A calculation procedure of general validity has been developed to unravel the effect of rapid exchange between the various species in equilibrium as a function of pD of the solution. The method of calculation used in this part of the new computer program, HYPNMR, is independent of the equilibrium model. HYPNMR has been used to obtain the basicity constants of spermine with respect to the D+ cation at 40 degrees C. Calculations have been performed using either 13C[1H] or 1H/13C data individually, or using both sets of data simultaneously. The results of the latter calculations were practically the same as the results obtained with the single data sets; the calculated errors on the refined parameters were a little smaller. After appropriate empirical corrections for temperature effects and for the presence of D+ in contrast to H+, the calculated constants are compared with spermine protonation constants which have been determined previously both from potentiometric and NMR data.

Carbon Isotopes↗
PubMed1995

Overproduction, purification and characterization of the cellulose-binding domain of the Erwinia chrysanthemi secreted endoglucanase EGZ.

EGZ is the major endoglucanase secreted by Erwinia chrysanthemi. Functional characterization indicates that it is made of a catalytic N-terminal domain linked to a C-terminal cellulose-binding domain (CBD) by a Ser/Thr-rich linker. A chimeric plasmid, in which the CBD-encoding region was fused downstream of the ompA signal sequence, was constructed and introduced into Escherichia coli. This allowed for the production of processed and disulfide-bonded CBD, mostly recovered from the culture supernatant of E. coli. One-dimensional NMR analysis of the purified CBD reveals that it folds into a well-structured domain. Moreover, comparison with the one-dimensional NMR analysis of full-length EGZ strongly suggests that the CBD folds autonomously, providing experimental support for the existence of domains of EGZ.

Binding Sites↗
PubMed1994

NMR structures of ferredoxin chloroplastic transit peptide from Chlamydomonas reinhardtii promoted by trifluoroethanol in aqueous solution.

The 32-amino acid transit peptide of the unicellular green alga Chlamydomonas reinhardtii ferredoxin has been synthesized and analysed by NMR spectroscopy and circular dichroism. The results show that while the peptide is unstructured in water, it undergoes an alpha-helix formation from residue 3 to 13 in a 30:70 molar-ratio mixture of 2,2,2-trifluoroethanol. The remainder of the peptide is still unstructured in CF3CD2OD/H2O mixtures, but is distributed on a side opposite to a hydrophobic ridge formed by Met5, Phe9 and Val13 on the induced alpha-helix. The NMR structures driven by 2,2,2-trifluoroethanol in aqueous solution, are discussed in terms of potent interactions with the chloroplast envelope and its translocation molecular machinery.

Amino Acid Sequence↗
PubMed1994

Spatio-temporal variations in population growth in India since 1901.

"On the basis of census data we analyse population growth since 1901 first for India as a whole, secondly on State level. The spatio-temporal patterns suggest a process of innovation and diffusion with regard to the spatial change of natural increase. The southern States cover more stages of the model of demographic transition than the northern States. Explanations about these spatial differentials come for example [from] sociocultural differences between north and south determining two demographic regions according to Satpura Range." (SUMMARY IN GER AND RUS)

Asia↗
PubMed1993

The problem of fuzzy cause-specific death rates in mortality context analysis: the case of Panama City.

In studies of mortality, small and fluctuating numbers of deaths are problems which are caused by infrequent reporting and small spatial unit reporting. To use Panama City as an example, the paper will introduce a Monte Carlo simulation which allows for the analysis of mortality even with small absolute numbers. In addition, Panama City will be used as an example where good medical care is available in every city district, so that social class differences between the districts have a negligible effect on most cause-specific death rates and infant mortality.

Adolescent↗
PubMed1991

Relationship between Respiration and Photosynthesis in Guard Cell and Mesophyll Cell Protoplasts of Commelina communis L.

A mass spectrometric method combining (16)O/(18)O and (12)C/(13)C isotopes was used to quantify the unidirectional fluxes of O(2) and CO(2) during a dark to light transition for guard cell protoplasts and mesophyll cell protoplasts of Commelina communis L. In darkness, O(2) uptake and CO(2) evolution were similar on a protein basis. Under light, guard cell protoplasts evolved O(2) (61 micromoles of O(2) per milligram of chlorophyll per hour) almost at the same rate as mesophyll cell protoplasts (73 micromoles of O(2) per milligram of chlorophyll per hour). However, carbon assimilation was totally different. In contrast with mesophyll cell protoplasts, guard cell protoplasts were able to fix CO(2) in darkness at a rate of 27 micromoles of CO(2) per milligram of chlorophyll per hour, which was increased by 50% in light. At the onset of light, a delay observed for guard cell protoplasts between O(2) evolution and CO(2) fixation and a time lag before the rate of saturation suggested a carbon metabolism based on phosphoenolpyruvate carboxylase activity. Under light, CO(2) evolution by guard cell protoplasts was sharply decreased (37%), while O(2) uptake was slowly inhibited (14%). A control of mitochondrial activity by guard cell chloroplasts under light via redox equivalents and ATP transfer in the cytosol is discussed. From this study on protoplasts, we conclude that the energy produced at the chloroplast level under light is not totally used for CO(2) assimilation and may be dissipated for other purposes such as ion uptake.

Journal Article↗