Structural and functional characterization of an egg-laying hormone signaling system in a lophotrochozoan - The pacific oyster (Crassostrea gigas).

The egg-laying hormones (ELHs) of gastropod mollusks were characterized more than forty years ago. Yet, they have remained little explored in other mollusks. To gain insights into the functionality of the ELH signaling system in a bivalve mollusk - the oyster Crassostrea gigas, this study investigates the processing of its ELH precursor (Cragi-ELH) by mass spectrometry. Some of the ELH mature peptides identified in this study were subsequently investigated by nuclear magnetic resonance and shown to adopt an extended alpha-helix structure in a micellar medium mimicking the plasma membrane. To further characterize the ELH signaling system in C. gigas, a G protein-coupled receptor phylogenetically related to ecdysozoan diuretic hormone DH44 and corticotropin-releasing hormone (CRH) receptors named Cragi-ELHR was also characterized functionally and shown to be specifically activated by the two predicted mature ELH peptides and their N-terminal fragments. Both Cragi-ELH and Cragi-ELHR encoding genes were mostly expressed in the visceral ganglia (VG). Cragi-ELH expression was significantly increased in the VG of both fully mature male and female oysters at the spawning stage. When the oysters were submitted to a nutritional or hyposaline stress, no change in the expression of the ligand or receptor genes was recorded, except for Cragi-ELHR only during a mild acclimation episode to brackish water. These results suggest a role of Cragi-ELH signaling in the regulation of reproduction but not in mediating the stress response in our experimental conditions.

1H NMR structural characterization of the cytochrome c modifications in a micellar environment.

The interaction of cytochrome c with micelles of sodium dodecyl sulfate was studied by proton NMR spectroscopy. The protein/micelles ratio was found to be crucial in controlling the extent of the conformational changes in the heme crevice. Over a range of ratios between 1:30 and 1:60, the NMR spectra of the ferric form display no paramagnetic signals due to a moderately fast exchange between intermediate species on the NMR time scale. This is consistent with an interconversion of bis-histidine derivatives (His18-Fe-His26 and His18-Fe-His33). Further addition of micelles induces a high-spin species that is proposed to involve pentacoordinated iron. The resulting free binding site, also encountered in the ferrous form, is used to complex exogenous ligands such as cyanide or carbon monoxide. Attribution of the heme methyls was performed by means of exchange spectroscopy through ligand exchange or electron transfer. The heme methyl shift pattern of the micellar cyanocytochrome in the ferric low spin form is different from the pattern of both the native and the cyanide cytochrome c adduct, in the absence of micelles, reflecting a complete change of the heme electronic structure. Analysis of the electron self-exchange reaction between the two redox states of the micellar cyanocytochrome c yields a rate constant of 2.4 x 10(4) M(-1) s(-1) at 298 K, which is surprisingly close to the value observed in the native protein.

Sarcolemma phospholipid structure investigated by immunogold electron microscopy and (31)P NMR spectroscopy with lanthanide ions.

The biological functions of plasma membranes depend greatly on the biophysical properties resulting from protein and phospholipid structure. We investigated the phospholipid structure of the normal sarcolemma membrane, which is known to be highly dysfunctional in myopathies. Combining electron microscopy and (31)P nuclear magnetic resonance (NMR) spectroscopy on isolated sarcolemma vesicles, we find that (i) the sarcolemma vesicles maintain the in-vivo cellular sidedness, (ii) the phospholipid mobility is close to that observed in model membranes (similar lateral diffusion coefficients and spin-lattice T(1) relaxation times). Using broad-band and magic angle spinning (31)P NMR spectroscopy with lanthanide ions (Pr(3+)), it is possible to quantify the distribution of phospholipids between internal and external membrane layers, showing that the trans-bilayer distribution is highly asymmetrical.

Determination of metabolic fluxes during glucose catabolism in Propionibacterium freudenreichii subsp. shermanii.

In vivo 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used to investigate the pathways of glucose metabolism, non-invasively, in living cell suspensions of Propionibacterium freudenreichii subsp. shermanii. This species is the main ripening flora of the Swiss-type cheeses and is widely used as propionic acid and vitamin B12 industrial producer. The flow of labelled [1-13C]glucose was monitored in living cell suspensions and enrichment was detected in main products like [1-13C]glycogen, [6-13C]lycogen, [1-13C]trehalose, [6-13C]trehalose, [1-13C]propionate, [2-13C]propionate, [3-13C]propionate, [1-13C]acetate, [2-13C]acetate, [1-13C]succinate, [2-13C]succinate and [1-13C]CO2. alpha and beta glucose consumption could be examined separately and were catabolized at the same rate. Three intermediates were also found out, namely [1-13C]glucose-6-phosphate, [6-13C]glucose-6-phosphate and [1-13C]glucose-1-phosphate. From the formation of intermediates such as [6-3C]glucose-6-phosphate and products like [6-13C]glycogen from [1-13C]glucose we concluded the bidirectionality of reactions in the first part of glycolysis and the isomerization at the triose-phosphate level. Comparison of spectra obtained after addition of [1-13C]glucose or [U-12C]glucose revealed production of [1-13C]CO2 which means that pentose phosphates pathway is active under our experimental conditions. From the isotopic pattern of trehalose, it could be postulated that trehalose biosynthesis occurred either by direct condensation of two glucose molecules or by gluconeogenesis. A chemically defined medium was elaborated for the study and trehalose was the main osmolyte found in the intracellular fraction of P. shermanii grown in this medium.

Assignment of heme methyl 1H-NMR resonances of high-spin and low-spin ferric complexes of cytochrome p450cam using one-dimensional and two-dimensional paramagnetic signals enhancement (PASE) magnetization transfer experiments.

An 1H-NMR study of ferric cytochrome P450cam in different paramagnetic states was performed. Assignment of three heme methyl resonances of the isocyanide adduct of cytochrome P450 in the ferric low-spin state was recently performed using electron exchange in the presence of putidaredoxin [Mouro, C., Bondon, A., Jung, C., Hui Bon Hoa, G., De Certaines, J.D., Spencer, R.G.S. & Simonneaux, G. (1999) FEBS Lett. 455, 302-306]. In this study, heme methyl protons of cytochrome P450 in the native high-spin and low-spin states were assigned through one-dimensional and two-dimensional magnetization transfer spectroscopy using the paramagnetic signals enhancement (PASE) method. The order of the methyl proton chemical shifts is inverted between high-spin and low-spin states. The methyl order observed in the ferric low-spin isocyanide complexes is related to the orientation of the cysteinate ligand.

Proton nuclear magnetic resonance study of the binary complex of cytochrome P450cam and putidaredoxin: interaction and electron transfer rate analysis.

A 1H nuclear magnetic resonance study of the complex of cytochrome P450cam-putidaredoxin has been performed. Isocyanide is bound to cytochrome P450cam in order to increase the stability of the protein both in the reduced and the oxidized state. Diprotein complex formation was detected through variation of the heme methyl proton resonances which have been assigned in the two redox states. The electron transfer rate at equilibrium was determinated by magnetization transfer experiments. The observed rate of oxidation of reduced cytochrome P450 by the oxidized putidaredoxin is 27 (+/- 7) per s.

PASE (PAramagnetic signals enhancement): a new method for NMR study of paramagnetic proteins.

A new method for NMR spectra acquisition of paramagnetic proteins is described, based on the simple use of homonuclear broadband decoupling of the diamagnetic region. Several advantages are associated with this method which was applied to one-dimensional spectra, to 1D NOE-difference spectroscopy, and to 2D NOESY. The main advantage is a very flat baseline obtained using the PASE (paramagnetic signals enhancement) method. Furthermore, the bulky region of the diamagnetic protons being suppressed, clean NOE-difference spectra can be acquired as well as improved 2D NOESY maps. Applications on 1D 1H spectrum of bovine liver catalase (MW 230,000), and 1D and 2D on the high-spin form of the myoglobin, used as a model protein, are presented.

1H-NMR study of diamagnetic cytochrome P450cam: assignment of heme resonances and substrate dependance of one cysteinate beta proton.

The 1H-NMR study of diamagnetic cytochrome P450cam FeII-CO has been performed for the first time. Chemical shifts of the cysteinate fifth ligand protons and of several heme protons have been assigned through 1- and 2-dimensional spectra at 500 MHz. A substrate dependance has been observed for the resonance of the cysteinate proton detected in the high-field region.

Proton and phosphorus nuclear magnetic resonance spectroscopy of human brain tumor extracts with automatic data classification: a preliminary study.

High-resolution one-dimensional proton and phosphorus and two dimensional COSY proton Magnetic Resonance Spectroscopy were used to investigate the lipid and carbohydrate metabolism of human brain tumors. Sixteen meningioma (MG) (benign tumors) and ten glioblastoma (GB) (malignant tumors) samples from brain surgery were treated for dual extraction of lipidic and aqueous phases before NMR processing. A highly significant variation of the 1H metabolite spectral pattern was observed between benign and malignant tumors. Double extraction method combined with both 1H and 31P NMR in vitro analyses provided a large set of biochemical information which may be statistically analyzed to elucidate tumor-specific biochemical pathways and to improve interpretation of in vivo spectra.

Comparative Fourier transform infrared studies of the secondary structure and the CO heme ligand environment in cytochrome P-450cam and cytochrome P-420cam.

For the first time, Fourier transform infrared spectroscopy has been applied to cytochrome P-450 to analyze the protein secondary structure. From Fourier self-deconvolution and fitting the infrared spectra in the amide I' region (1600-1700 cm-1), we estimate 44% alpha-helix, 31% beta-sheet, and 18% turns for substrate-free cytochrome P-450cam. In the presence of camphor, 54% alpha-helix and 310-helix, 21% beta-sheet, and 21% turns are obtained which agree with the crystallographic data of 53% alpha-helix, 19% beta-sheet, and 16% turns [Poulos, T. L., Finzel, B. C., & Howard, A. J. (1987) J. Mol. Biol. 195, 687-700]. Cytochrome P-420cam is produced from substrate-free cytochrome P-450cam in two ways: (i) by temperature elevation up to 60 degrees C and (ii) by exposure to KSCN up to 1.5 M. The secondary structure composition is determined for each temperature and KSCN concentration and compared with the changes observed in the iron ligand CO stretch vibration bands appearing between 1900 and 2000 cm-1. Thermally induced cytochrome P-420 has an alpha-helix content of 19%, a beta-sheet content of 53%, 14% turns, and 5% antiparallel beta-sheets from intermolecular hydrogen bonds within protein aggregates. The formation of cytochrome P-420 as a function of the KSCN concentration indicates two types of cytochrome P-420. Up to 1 M KSCN, the induced cytochrome P-420 displays only little modification of the secondary structure, whereas at 1.5 M KSCN, larger changes are observed, resulting in 85% cytochrome P-420 without protein precipitation and containing 30% alpha-helix, 48% beta-sheet, and 17% turns. Infrared spectra in the iron ligand CO stretch region show several subconformers for cytochrome P-420. During the cytochrome P-420 formation, the CO stretch modes are shifted to higher frequencies by 3-11 cm-1, with a main feature at about 1964 cm-1, compared to those of substrate-free cytochrome P-450cam-CO.

Substrate interactions in cytochrome P-450: correlation between carbon-13 nuclear magnetic resonance chemical shifts and C-O vibrational frequencies.

13CO NMR chemical shifts and 12CO infrared stretching frequencies have been measured for cytochrome P-450cam-CO in the presence of D-camphor and of various camphor analogues. A linear correlation between both parameters delta (13C) and v(CO) was found indicating that the steric and electrostatic interactions acting on the CO ligand are influenced by the substrate. It has been proved that P-450 complexes are on another line in this correlation than hemoglobins which is explained by the different proximal ligand.

Solution structure determination by NMR spectroscopy of a synthetic peptide corresponding to a putative amphipathic alpha-helix of spiralin: resonance assignment, distance geometry and simulated annealing.

Spiralin is the major protein of the plasma membrane of several spiroplasmas. Neither the function of this protein nor the crystallographic structure is known. Analysis of the primary structure of spiralin from Spiroplasma melliferum BC3 suggests the presence of an amphipathic peptide in the 143-162 region (Chevalier, C., Saillard, C. and Bové, J.M. (1990) J. Bacteriol. 172, 6090-6097). The structure of a synthetic peptide, H2N-L-N-A-V-N-T-Y-A-T-L-A-K-A-V-L-D-A-I-Q-N-amide, corresponding to this fragment has been examined by 1H-NMR spectroscopy. This 20 amino acid peptide adopts a random coil structure in solution, but the addition of trifluoroethanol stabilizes a structure exhibiting alpha-helical character. The 1H-NMR spectrum has been fully assigned in CF3CD2OD/H2O (30:70, v/v) and the three-dimensional structure has been elucidated using NMR-derived distance information. The calculated structures have been obtained by dynamical simulated annealing or distance geometry followed by simulated annealing. Both sets of structures have been energy-minimized using CHARMm potential. The resulting structures are very similar in terms of constraint violations and energies. It is demonstrated that whereas the first three residues exhibit a large flexibility, the remaining sequence is helical.

Trimethylphosphine binding to horse-heart and sperm-whale myoglobins. Kinetics, proton magnetic resonance assignment and nuclear Overhauser effect investigation of the heme pocket.

Two-dimensional nuclear magnetic resonance techniques have been used to assign resonances corresponding to the heme pocket and several other residues of horse heart and sperm whale myoglobins ligated by trimethylphosphine. The assignment procedure was based mainly on the nuclear Overhauser effect connectivities with the ligand and the heme substituents. For quantitative measurements of Overhauser effects, application of truncated driven techniques between a proton from distal residues and methyl groups from the ligand was used to determine internuclear distances. These new results have permitted us to map the heme pockets and to investigate the conformational differences in the heme pockets between horse heart and sperm whale myoglobins. The interproton distances between distal amino acid residues and trimethylphosphine were found to be longer in horse heart myoglobin relative to those in sperm whale myoglobin. This result suggests that the size of the heme pocket is larger in horse heart myoglobin. Association and dissociation rate constants were measured for trimethylphosphine binding to myoglobins. Both values were four times larger for horse heart myoglobin than those for sperm whale myoglobin. This observation confirms the structural results obtained with NMR studies and is rationalized by a greater stabilization of a larger pocket in horse heart myoglobin relative to sperm whale myoglobin.

Electron-transfer self-exchange kinetics of trimethylphosphine horse-heart myoglobin.

Electron self-exchange has been measured by an NMR technique for horse-heart myoglobin. The rate is 3.1 x 10(3) M-1 s-1 at 23 degrees in 0.1 M phosphate at pH 6.9. The rate was weakly dependent on ionic strength up to 0.7 M in added KCl (3.9 x 10(3) M-1 s-1). The enthalpy of activation was 12.1 +/- 0.5 kcal mol-1, and the entropy of activation was -1.2 +/- 0.5 cal mol-1 deg-1. Analysis of the data in terms of the Marcus theory gives a reorganization energy, lambda, for self-exchange of 1.6 eV mol-1.

A possible calcium binding site in animal lectins: a 1H-NMR study of the interaction between lanthanides and a synthetic peptide from a highly conserved domain of Pleurodeles lectin.

1H-NMR techniques have been used to study the metal binding properties of a synthetic peptide of 15 amino acids corresponding to a highly conserved domain of Pleurodeles lectin. The addition of lanthanum chloride or praseodymium chloride in a peptide solution induces some conformational changes as displayed by several concerted variations of peptide resonances. The Ln3+ concentration dependence of the chemical shifts was used to calculate the Ln3+ binding constants. The dissociation constants of 95 microM and 280 microM were found for La3+ and Pr3+, respectively.

31P-NMR investigation of trimethylphosphine binding to [alpha Fe(II), beta Mn(II)] hybrid hemoglobin. A model for partially liganded species.

31P-NMR of trimethylphosphine binding to the ferrous chains of a ([alpha Fe(II), beta Mn(II)]hemoglobin hybrid is employed to investigate partially liganded species. This study shows that at low pH (6.5), in the presence of inositol hexaphosphate, the resonance at 23.2 ppm (from H3PO4) is due to phosphine bonding to alpha-chains in the T quaternary state. At elevated pH (7.6), phosphine binding to the alpha-chains produces a resonance at 24.8 ppm which is associated with a T-to-R conversion. These findings are discussed in relation with our previous results on direct observation of intermediate ligation states of hemoglobin.

Proton hyperfine resonance assignments in trimethylphosphine ligated ferrimyoglobin using saturation transfer spectroscopy.

1H-NMR saturation transfer spectroscopy and nuclear Overhauser effect (NOE) have been utilized to assign several heme resonances in the low-spin trimethyl phosphine complex of sperm whale metmyoglobin. The two methods permit the location of the heme methyl resonances and the heme 2-vinyl group resonances. A qualitative comparison involving the methyl shift pattern in metMbN3, metMbCN, imidazole metMb and trimethyl phosphine metMb shows a reverse methyl shift between pyrrole I and pyrrole IV. The different hyperfine shift pattern for metMbPMe3 is suggested to arise from: (i) a possible reorientation of the proximal histidine plane; (ii) different heme protein contacts in the different ligated proteins, and (iii) a small contribution from high-spin character. The 2-vinyl group is formed in the cis plane orientation.

Oxidation of cycloalkylamines by cytochrome P-450. Mechanism-based inactivation, adduct formation, ring expansion, and nitrone formation.

Mechanism-based destruction of cytochrome P-450 (P-450) and P-450 heme is observed during the oxidation of N-cyclopropyl and N-cyclobutyl benzylamines. The slower inactivation by the cyclobutylamines relative to cyclopropylamines is consistent with known relative rates of ring opening of cycloalkyl-substituted aminium radicals. Evidence was found that porphyrin meso adducts of the type reported for horseradish peroxidase and cyclopropanone hydrate (Wiseman, J. S., Nichols, J. S., and Kolpak, M. X. (1982) J. Biol. Chem. 257, 6328-6332) were not formed. Radiolabels from cyclopropylamine substrates were covalently attached to protein but essentially only from the cyclopropyl portion and not the benzylic portion. Neither label appeared to be bound to extractable heme; however, during oxidations with cyclopropylamines, labeled P-450 heme became covalently attached to protein. Oxidation of 1-phenylcyclobutylamine by P-450 yielded 2-phenyl-1-pyrroline and 2-phenylpyrrolidine, and the ring expansion is interpreted as evidence for the existence of aminium radicals based on precedents with monoamine oxidase (Silverman, R. B., and Zieske, P. A. (1985) Biochemistry 24, 2128-2138). In addition, purified P-450PB-B oxidized N-(1-phenylcyclobutyl)-benzylamine to N-(1-phenyl)cyclobutyl phenyl nitrone, identified using spectral techniques. This transformation involves two sequential oxidations with either a hydroxylamine or benzylidene intermediate. While P-450 oxidized the amine to both compounds, only the hydroxylamine was rapidly oxidized to give the nitrone. The ring expansion and nitrone products are interpreted in the context of aminium radical intermediates involved in the mechanism of P-450-catalyzed amine oxidation.

Phosphines as a new structural probe of hemoglobin. 1H-NMR evidence for perturbations in the beta heme pocket induced by a thiol reagent.

Binding of trimethylphosphine to myoglobins and hemoglobins from a variety of sources has been examined by 1H-nuclear magnetic resonance. The hemoglobins exhibit two resonances at high field (approx. -3.5 ppm) which have been assigned to PMe3 bound to alpha or to beta subunits. Perturbations in the beta heme pocket induced by a thiol reagent have been detected both in 1H and 31P spectra.