SOLEIL shining on the solution-state structure of biomacromolecules by synchrotron X-ray footprinting at the Metrology beamline.

Synchrotron X-ray footprinting complements the techniques commonly used to define the structure of molecules such as crystallography, small-angle X-ray scattering and nuclear magnetic resonance. It is remarkably useful in probing the structure and interactions of proteins with lipids, nucleic acids or with other proteins in solution, often better reflecting the in vivo state dynamics. To date, most X-ray footprinting studies have been carried out at the National Synchrotron Light Source, USA, and at the European Synchrotron Radiation Facility in Grenoble, France. This work presents X-ray footprinting of biomolecules performed for the first time at the X-ray Metrology beamline at the SOLEIL synchrotron radiation source. The installation at this beamline of a stopped-flow apparatus for sample delivery, an irradiation capillary and an automatic sample collector enabled the X-ray footprinting study of the structure of the soluble protein factor H (FH) from the human complement system as well as of the lipid-associated hydrophobic protein S3 oleosin from plant seed. Mass spectrometry analysis showed that the structural integrity of both proteins was not affected by the short exposition to the oxygen radicals produced during the irradiation. Irradiated molecules were subsequently analysed using high-resolution mass spectrometry to identify and locate oxidized amino acids. Moreover, the analyses of FH in its free state and in complex with complement C3b protein have allowed us to create a map of reactive solvent-exposed residues on the surface of FH and to observe the changes in oxidation of FH residues upon C3b binding. Studies of the solvent accessibility of the S3 oleosin show that X-ray footprinting offers also a unique approach to studying the structure of proteins embedded within membranes or lipid bodies. All the biomolecular applications reported herein demonstrate that the Metrology beamline at SOLEIL can be successfully used for synchrotron X-ray footprinting of biomolecules.

At5g50600 encodes a member of the short-chain dehydrogenase reductase superfamily with 11beta- and 17beta-hydroxysteroid dehydrogenase activities associated with Arabidopsis thaliana seed oil bodies.

In a previous work, we presented evidence for the presence of a protein encoded by At5g50600 in oil bodies (OBs) from Arabidopsis thaliana [P. Jolivet, E. Roux, S. D'Andrea, M. Davanture, L. Negroni, M. Zivy, T. Chardot, Protein composition of oil bodies in Arabidopsis thaliana ecotype WS, Plant Physiol. Biochem. 42 (2004) 501-509]. Using specific antibodies and proteomic techniques, we presently confirm the existence of this protein, which is a member of the short-chain steroid dehydrogenase reductase superfamily. We have measured its activity toward various steroids (cholesterol, dehydroepiandrosterone, cortisol, corticosterone, estradiol, estrone) and NAD(P)(H), either within purified OBs or as a purified bacterially expressed chimera. Both enzymatic systems (OBs purified from A. thaliana seeds as well as the chimeric enzyme) exhibited hydroxysteroid dehydrogenase (HSD) activity toward estradiol (17beta-hydroxysteroid) with NAD+ or NADP+, NADP+ being the preferred cofactor. Low levels of activity were observed with cortisol or corticosterone (11beta-hydroxysteroids), but neither cholesterol nor DHEA (3beta-hydroxysteroids) were substrates, whatever the cofactor used. Similar activity profiles were found for both enzyme sources. Purified OBs were found to be also able to catalyze estrone reduction (17beta-ketosteroid reductase activity) with NADPH. The enzyme occurring in A. thaliana OBs can be classified as a NADP+-dependent 11beta-,17beta-hydroxysteroid dehydrogenase/17beta-ketosteroid reductase. This enzyme probably corresponds to AtHSD1, which is encoded by At5g50600. However, its physiological role and substrates still remain to be determined.

Protein components of low-density lipoproteins purified from hen egg yolk.

To identify apoproteins present in purified low-density lipoproteins from hen egg yolk in relation with their emulsifying properties, they have been separated by SDS-PAGE. We identified two different proteins by liquid chromatography-tandem mass spectrometry analysis of the peptides obtained by the trypsin digestion of protein gel bands. Apovitellenin I was identified as a monomer and a dimer. Its amino acid sequence was totally confirmed, and molecular mass determination by liquid chromatography-mass spectrometry showed that it did not present post-translational modifications but only a slight heterogeneity by the loss of one or two amino acids at the C-terminal part of the protein. Apolipoprotein B was identified into seven bands corresponding to fragments resulting of a processing of the hen blood apo-B protein. The identity of the fragments was determined by the observation of the sequence coverage by trypsin peptides and the sequence alignment with homologous human blood apolipoprotein B-100.

The effect of phosphate on the unfolding-refolding of phosphoglycerate kinase induced by guanidine hydrochloride.

Phosphate ions were found to stabilize the native structure of phosphoglycerate kinase without modifying the folding pathway. The transition curves obtained from different signals: enzyme activity, ellipticity at 220 nm and fluorescence intensity at 336 nm (excitation at 292 nm) are shifted to smaller guanidine hydrochloride cm values in the absence of phosphate. The kinetic characteristics are qualitatively similar, unfolding rate constants being slightly smaller in the presence of phosphate. The mechanism by which the native structure of phosphoglycerate kinase is stabilized by phosphate probably occurs upon specific phosphate binding to the nucleotide beta- or gamma-phosphate binding site of nucleotides.

Non reductive activation of spinach chloroplastic fructose-1,6-bisphosphatase: evidence for structural modification of the enzyme.

Preincubation of chloroplastic fructose-1,6-bisphosphatase (FBPase) in the presence of Ca2+/fructose-1,6-bisphosphate (FBS) gives rise to an active enzyme. This non-reductive activation at pH 8 occurs in the same range of time (min) as the well known reductive activation by thioredoxins and this process is reversible. A conformational change of the enzyme occurs upon the activation by Ca2+/FBP. Indeed, the circular dichroism and the fluorescence spectra of the inactive and active enzymes are different. The titration of the sulfhydryl groups of both enzymes indicates that one -SH group per monomer is unmasked upon activation, and the isoelectrofocusing pattern shows that the pI of inactive FBPase is shifted from 4.26 to 4.56 upon this non-reductive process.

Overphosphorylation of milk caseins by a recombinant protein kinase CK2 catalytic subunit.

Milk caseins have been phosphorylated by a recombinant protein kinase CK2 catalytic subunit from Schizosaccharomyces pombe (rspCK2alpha). Phosphate incorporation stoechiometries into purified caseins and into native phosphocaseinate, a substrate exhibiting a micellar-like structure, were determined. We incorporated 2.01 mol of P/mol of alpha-casein, 6.46 mol of P/mol of beta-casein, up to 0. 29 mol of P/mol of kappa-casein in 4 h, and more than 1.36 mol of P/mol of casein into phosphocaseinate under optimized conditions. Phosphocaseinate was sequentially phosphorylated; beta-caseins being labeled at first; alpha-caseins being labeled later; and to a lower extend, kappa-caseins were the last to be phosphorylated. The solubility of phosphocaseinate micelles increased by 1.34 from 65 to 87%, and its rennetting time was increased 2.88 times. These results are discussed in terms of plausible structural organization of caseins micelles and the effect of phosphorylation on their structure.

Regulation of Crassula argentea phosphoenolpyruvate carboxylase in relation to temperature.

The effect of temperature on the kinetic parameters of phosphoenolpyruvate carboxylase purified from Crassula argentea was such that both the Vmax and Km(MgPEP) values tended upward over the range from 11 to 35 degrees C. The increased rate at low temperatures due to the low Km is at least partially offset by the increased Vmax at higher temperatures, potentially leading to a broad plateau of enzyme activity and a relatively small effect of temperature on the enzyme. The cooperativity was negative at 11 degrees C, but above 15 degrees C it became positive. The presence of 5 mM glucose-6-phosphate has relatively little effect on Vmax but it clearly reduces Km and overcomes any effect of temperature on this parameter in the range studied. Positive cooperativity is observed only at temperatures above 25 degrees C. The size of the native enzyme, as determined by dynamic light scattering, was strongly toward the tetrameric form. At a temperature of 40 degrees C and above, a considerable oligomerization takes place. No loss of activity can be observed in this range of temperature. In the presence of either glucose-6-phosphate or magnesium phosphoenolpyruvate, at temperatures under 25 degrees C, the equilibrium is displaced toward higher levels of aggregation. Maximal accumulation of lead malate occurred at 10 to 12 degrees C in vivo with reduction to about 25% at 35 degrees C. Glucose-6-phosphate followed a similar curve in response to temperature, but the overall difference was about 50%. The sum of phosphoenolpyruvate plus pyruvate is level at night temperatures below 25 degrees C, doubling at 35 degrees C. Calculated concentrations of malate, glucose-6-phosphate, and phosphoenolpyruvate plus pyruvate indicate that the concentrations present are equal to or greater than Ki, Ka, and Km values for these metabolites, respectively.

Role of cysteine in activation and allosteric regulation of maize phosphoenolpyruvate carboxylase.

The effect of 5-5'-dithiobis-2-nitrobenzoate (DTNB) on the kinetic parameters and structure of phosphoenolpyruvate carboxylase purified from maize (Zea mays L.) has been studied. The V(max) is found to be independent of the presence of this thiol reagent. The K(m) is increased upon oxidation of cysteines by DTNB. At a substrate concentration higher than K(m) (3.1 millimolar Mgphosphoenolpyruvate), a significant reversible decrease of the activity is observed. Malate has little effect in preventing the modification of these cysteines. The V type inhibition by malate was also studied at a saturating phosphoenolpyruvate level (9.3 millimolar Mgphosphoenolpyruvate). In the presence of 50 micromolar DTNB, up to 60% inhibition is caused by 15 millimolar malate; however, in the presence of both 50 micromolar DTNB and 50 millimolar dithiothreitol (DTT) this inhibition is reduced to 20%. The presence of DTT alone increases the size of the phosphoenolpyruvate carboxylase molecule as determined by light scattering. The activity at nonsaturating substrate concentration is increased by 36% in the presence of DTT. The oligomerization equilibrium between the dimer and the tetrameric form of the enzyme is affected by cysteine. The K(m) for the substrate, the sensitivity toward malate, and the size of the enzyme are found to be modified upon incubation in the presence of DTT.

Properties of oxidized and reduced spinach (Spinacia oleracea) chloroplast fructose-1,6-bisphosphatase activated by various agents.

Fructose-1,6-bisphosphatase (FBPase) can be reduced and activated by either dithiothreitol or reduced thioredoxin. This activation is pH-dependent. An amino acid group with a pK value of 5.55 is involved in this process. Both enzyme forms can also be stimulated by agents such as fructose 1,6-bisphosphate, Mg2+, Ca2+ and Ca2+/fructose 1,6-bisphosphate. FBPase reduced by dithiothreitol is more strongly activated than the enzyme reduced by thioredoxin. The specificity constant (kcat./Km) is enhanced over 2.5-25-fold and 1.5-2-fold (depending on the agent used) for FBPase reduced by dithiothreitol and thioredoxin respectively. In both cases, no new kinetic properties appeared. The pH-activity profile of the stimulated enzyme is slightly shifted towards the acidic side with respect to the reduced enzyme. A lag phase is observed in the progress curve of both enzymic forms, treated or untreated. Each agent used to stimulate must induce a new conformation of the enzyme, more active than the initial one, characterized by a specificity constant and a relaxation time. This lag phase tends to disappear when the assay temperature is increased. Temperature has the same effect on the activity of oxidized, reduced and stimulated FBPase, but different effects on the stability of the different forms.

Dual specificity of casein kinase II from the yeast Yarrowia lipolytica.

In the case of protein kinases, and especially in the case of casein kinase II (CKII), a link has been found between the type of the amino acids autophosphorylated and the targeted amino acids on the substrates. In the presence of Mg2+, CKII from the yeast Yarrowia lipolytica is autophosphorylated on serines and threonines, and a serine threonine kinase activity is found predominantly when casein is used as substrate. In the presence of Mn2+, CKII autophosphorylation is inhibited on serines, and autophosphorylation on tyrosines, negligible in the former case, becomes significant. Tyrosine phosphorylation is then found to occur on casein. Mn2+ transforms CKII into a protein kinase with dual specificity, shifting its specificity from serine/threonine kinase towards a serine/threonine and tyrosine kinase. Mn2+ decreases the level of serine and threonine phosphorylation observed, while on the other hand promoting tyrosine kinase activity.

Inactivation of maize phosphoenolpyruvate carboxylase by urea.

Phosphoenolpyruvate carboxylase purified from leaves of maize (Zea mays, L.) is sensitive to the presence of urea. Exposure to 2.5 m urea for 30 min completely inactivates the enzyme, whereas for a concentration of 1.5 m urea, about 1 h is required. Malate appears to have no effect on inactivation by urea of phosphoenolpyruvate carboxylase. However, the presence of 20 mm phosphoenolpyruvate or 20 mm glucose-6-phosphate prevents significant inactivation by 1.5 m urea for at least 1 h. The inactivation by urea is reversible by dilution. The inhibition by urea and the protective effects of phosphoenolpyruvate and glucose-6-phosphate are associated with changes in aggregation state.

A protein kinase is located in the micellar fraction of fresh pasteurized skimmed farm milk.

Recombinant protein kinase CK2 from the yeast Schizosaccharomyces pombe is able to phosphorylate casein in skimmed pasteurized milk. We could incorporate up to 540 pmol of phosphate into 50 microg milk proteins, i.e., 0.26 P/mol caseins. To better understand the action of protein kinase CK2 on milk proteins, we have compared the action of rspCK2alpha on milk, and on different casein micellar subfractions isolated from milk by ultracentrifugation. In contrast to the situation observed with phosphocaseinate, alpha(s) casein was the best substrate for rspCK2alpha, whether milk or micellar fractions were used as substrates. We have characterized the protein content of different micellar fractions obtained by ultracentrifugation of cow milk using capillary zone electrophoresis. We confirm that the kappa casein content of micelles largely decreases when their size increases. In contrast, the alpha(s) casein content slightly increased with micelles size and beta casein content remained constant. All of the micellar fractions were substrates for rspCK2alpha, but a significant amount of intrinsic protein kinase activity was also found. The intrinsic protein kinase used added ATP as phosphate donor, and was only slightly sensitive to high heparin concentration. It could phosphorylate micellar casein in milk ultrafiltrate, in the absence of addition of any metallic cofactor. Its activity was only slightly affected by the addition of either MgCl2 or MnCl2. CaCl2 activated the enzyme significantly. The intrinsic kinase lost its activity with time, and could incorporate from 9 to 26% of the total phosphate incorporated in the presence of rspCK2a. Alpha(s) casein was the best substrate of the intrinsic kinase, followed by beta casein. In the presence of CaCl2, the intrinsic kinase was found to incorporate up to 470 pmol of phosphate into 50 microg of milk proteins.

Time-co-ordinated control of glycogen synthase, protein phosphatase 2A and protein kinase CK2 during culture growth in Yarrowia lipolytica in relation to glycogen metabolism.

In the growth course of the lipolytic yeast Yarrowia lipolytica, the activities of protein phosphatase 2A (PP2A) and glycogen synthase (GS) rise during the exponential phase and concomitantly glycogen storage occurs in the cells. There is also an increase in the independence ratio (RI) indicating a shift from an inactive phosphorylated GS form to an active dephosphorylated GS form. During the early stationary phase, an increase in protein kinase CK2 (CK2) activity, a reversion of RI variation and a glycogen content decrease are observed. GS activity proved to be a good indicator of early culture growth phase. Experiments carried out with enzymes purified from Y. lipolytica show strong RI variations upon the action of CK2 and PP2Ac, and 32P incorporation into GS protein through phosphorylation by CK2. GS activity would be controlled by the sequential action of PP2A and CK2.

Mutation of recombinant catalytic subunit alpha of the protein kinase CK2 that affects catalytic efficiency and specificity.

In order to understand better the structural and functional relations between protein kinase CK2 catalytic subunit, the triphosphate moiety of ATP, the catalytic metal and the peptidic substrate, we built a structural model of Yarrowia lipolytica protein kinase CK2 catalytic subunit using the recently solved three-dimensional structure of the maize enzyme and the structure of cAMP-dependent protein kinase peptidic inhibitor (1CDK) as templates. The overall structure of the catalytic subunit is close to the structure solved by Niefind et al. It comprises two lobes, which move relative to each other. The peptide used as substrate is tightly bound to the enzyme, at specific locations. Molecular dynamic calculations in combination with the study of the structural model led us to identify amino acid residues close to the triphosphate moiety of ATP and a residue sufficiently far from the peptide that could be mutated so as to modify the specificity of the enzyme. Site-directed mutagenesis was used to replace by charged residues both glycine-48, a residue located within the glycine-rich loop, involved in binding of ATP phosphate moiety, and glycine-177, a residue close to the active site. Kinetic properties of purified wild-type and mutated subunits were studied with respect to ATP, MgCl(2) and protein kinase CK2 specific peptide substrates. The catalytic efficiency of the G48D mutant increased by factors of 4 for ATP and 17.5 for the RRRADDSDDDDD peptide. The mutant G48K had a low activity with ATP and no detectable activity with peptide substrates and was also inhibited by magnesium. An increased velocity of ADP release by G48D and the building of an electrostatic barrier between ATP and the peptidic substrate in G48K could explain these results. The kinetic properties of the mutant G177K with ATP were not affected, but the catalytic efficiency for the RRRADDSDDDDD substrate increased sixfold. Lysine 177 could interact with the lysine-rich cluster involved in the specificity of protein kinase CK2 towards acidic substrate, thereby increasing its activity.

Cloning of the gene encoding the catalytic subunit of casein kinase II from the yeast Yarrowia lipolytica.

Casein kinase II from the yeast Yarrowia lipolytica is a heterotetramer of the form alpha alpha' beta 2. We report on the cloning and sequencing of a partial cDNA and of the complete genomic DNA coding for the catalytic alpha subunit of the casein kinase II from this yeast species. The sequence of the gene coding for this enzyme has been analyzed. No intron was found in the gene, which is present in a single copy. The deduced amino acid sequence of the gene shows high similarity with those of alpha subunit described in other species, although, uniquely, Y. lipolytica CKII alpha lacks cysteines. We find that the alpha subunit sequence of Y. lipolytica CKII is shown greater homology with the corresponding protein from S. pombe than with that from S. cerevisiae. We have analyzed CKII alpha expression and CKII alpha activity. We show that expression of this enzyme is regulated. The catalytic subunit is translated from a single mRNA, and the enzyme is present at a very low level in Y. lipolytica, as in other yeasts.

Purification and characterization of the recombinant form of Acyl CoA oxidase 3 from the yeast Yarrowia lipolytica.

The Acyl CoA dependent oxidase 3 (Aox3p) from the yeast Yarrowia lipolytica, expressed in Escherichia coli, as an active protein with a 6 His tag at its N-terminal region has been purified to electrophoretic homogeneity. The purified enzyme exhibits a specific activity of 1.95 microM/min/mg using hexanoyl-CoA as substrate, and it remains active for at least 1 month upon storage at -30 degrees C in the presence of 35% (V/V) glycerol. The pH and temperature optima of the enzyme are 7.4 and 28-38 degrees C, respectively. Aox3p catalyzes the oxidation of both aliphatic acyl-CoA substrates of different chain lengths (e.g., hexanoyl-CoA, decanoyl-CoA, myristyl-CoA) as well as of the aromatic/heterocyclic ring-substituted chromogenic substrates, such as furylpropionyl-CoA. Of the above substrates, the efficiency of the enzyme, as judged by its kcat to Km ratio, exhibits the following order: decanoyl CoA > myristyl CoA > hexanoyl CoA > furyl-propionyl-CoA (FPCoA). Phenol, which is normally used in the coupled assay system for monitoring the H2O2 formation, functions as both an activator (at low concentrations) and a competitive inhibitor (at high concentrations) with respect to acyl-CoA substrates. The magnitude of activation and inhibition of the enzyme is dependent on the nature of the acyl-CoA substrates.

Expression and characterization of the recombinant catalytic subunit of casein kinase II from the yeast Yarrowia lipolytica in Escherichia coli.

The alpha catalytic subunit of casein kinase II from the yeast Yarrowia lipolytica has been cloned and overexpressed using the pT7-7 expression vector in Escherichia coli. Casein kinase activity is found in the bacterial extracts. The catalytic subunit is partially expressed in a soluble and active form, which is purified to electrophoretic homogeneity. Most of the enzyme was found in inclusion bodies. In this form, the enzyme, which is almost pure, exhibits a low specific activity. We have focused our efforts on methods to activate the protein from the inclusion bodies. We have studied the renaturation of urea-denaturated CKII catalytic subunit. We have tried different renaturation buffers and found that renaturation by dilution was more efficient than renaturation by dialysis. Treatment of the enzyme found in the inclusion bodies with different nondetergent sulfobetaines (NDSB) led to a time-dependent activation. NDSB195 is a V-type activator of the recombinant catalytic subunit of casein kinase II. The NDSB195-activated enzyme remained active at the room temperature for weeks. Kinetic properties of the recombinant casein kinase II subunit are similar to those of the purified holoenzyme (low Km for ATP and inhibition by heparin). Kinetic study indicates that the beta subunit could interact with the alpha subunit at the level of the catalytic site to enhance activity and to modify the kinetic behavior of the enzyme.

Enzymatic phosphorylation of food proteins by purified and recombinant protein kinase CK2.

Protein kinase CK2 formerly called casein kinase II is a protein kinase able to phosphorylate more than 100 proteic substrates. We have purified protein kinase CK2 from the yeast Y. lipolytica to phosphorylate milk and plant reserve proteins to a significant extent. In the case of plant reserve proteins, which are polymeric substrates, not all subunits are substrate for protein kinase CK2, even if non phosphorylated subunits contain significant potent phosphorylations sites. Best substrates were soy beta-conglycinin (0.72 P/mol) and dephosphorylated caseins (0.5 P/mol). We have studied some functional properties of phosphorylated caseins. Solubility was improved for all pH values but pI. Sensitivity to calcium has also been assessed, and it is slightly improved upon phosphorylation. We have cloned the catalytic subunit of protein kinase CK2 from yeast Y. lipolytica. The recombinant catalytic subunit expressed in E. coli was active and displayed kinetic properties similar to those of the purified enzyme. The recombinant catalytic subunit was able to phosphorylate plant reserve proteins and milk proteins to a significant extent. Best substrates were soy beta-conglycinin (1.0 P/mol), and glycinin (0.59 P/mol).