Papers by Philippe Champeil
ATPase activities of various streptavidin-purified samples
PLOS ONE, 2014
<p><b>(A)</b> Coomassie Blue staining after SDS-PAGE of purified wild-type comp... more <p><b>(A)</b> Coomassie Blue staining after SDS-PAGE of purified wild-type complex (D<sup>WT</sup>-C), D560N variant (D<sup>D560N</sup>-C), E342Q variant (D<sup>E342Q</sup>-C), and wild-type Drs2p expressed alone. <b>(B–C and E–F</b>) The ATPase activity of the same samples (after 5-fold dilution resulting in about 60 µg/mL Drs2p in the case of the WT enzyme) was measured at 30°C in a KNG medium supplemented with 1 mg/mL DDM, 0.025 mg/mL PS and 1 mM Mg-ATP, in the absence (circles and dashed lines) or presence (triangles and continuous lines) of 0.025 mg/mL PI4P. The dotted line in panels C-F is given for easier comparison with results for WT. <b>(D)</b> Functional complementation of the temperature-sensitive phenotype of <i>Δdrs2</i> yeast cells. Yeast cells, either wild-type or <i>Δdrs2</i>, were transformed with plasmids bearing <i>DRS2</i> tagged at its 5′ end, either WT or E342Q. Cells transformed with an empty vector (EV) were used as negative control. Serial dilutions of yeast cells were spotted on plates and incubated at the restrictive temperature of 20°C.</p
Journal of Biological Chemistry, Apr 1, 2012
Background: Transport of phosphatidylserine (PS) analogs by the Drs2p flippase is regulated by Pt... more Background: Transport of phosphatidylserine (PS) analogs by the Drs2p flippase is regulated by PtdIns(4)P. Results: PS stimulates dephosphorylation of the Drs2p⅐Cdc50p complex only in the presence of PtdIns(4)P. Conclusion: The step at which PtdIns(4)P regulates lipid transport is identified. Significance: Our coordinated overexpression system provides mechanistic insight into PS transport and will be useful for further Drs2p characterization and crystallization.
The Topology of Sarcoplasmic Reticulum Ca2+-ATPase and Na+,K+-ATPase in the M5/M6 Region
Steinkopff eBooks, 1994
While there is general agreement concerning the existence of 4 hydrophobic membrane traverses in ... more While there is general agreement concerning the existence of 4 hydrophobic membrane traverses in the N-terminal part of P-type ATPases, the exact topology of the C-terminal, membraneous domain is still a matter of dispute. For sarcoplasmic reticulum (SR) Ca2+-ATPase 3 pairs of transmembrane helices (M5-M10) were proposed, leading to a 10-helical model for this ATPase, cf. Fig. 1. For Na+,K+-ATPase fewer membrane traverses were considered probable, leading to 7 helical (8) or 8 helical (3) models. In the 7 helical model, regions corresponding to M6, M8, and M10 were placed outside the lipid membrane, based on protein-chemical and immunochemical evidence. However, since there is strong evidence for cytosolic exposure of both the N-terminus and C-terminus, 8 helical models are now favored for Na+,K+-ATPase, but exactly how the transmembrane segments would be positioned in the membrane remains undefined. Concerning the different models proposed for Ca2+-ATPase and Na+,K+-ATPase it also needs to be asked, if it is plausible that their topology should be different, considering the fact that the hydropathic profiles of the two enzymes are strikingly similar.
Biochemistry, Sep 25, 1984
Journal of Biological Chemistry, Oct 1, 2002
After treatment of sarcoplasmic reticulum Ca 2؉-ATPase with proteinase K (PK) in the presence of ... more After treatment of sarcoplasmic reticulum Ca 2؉-ATPase with proteinase K (PK) in the presence of Ca 2؉ and a protecting non-phosphorylated ligand (e.g. adenosine 5-(,␥-methylenetriphosphate), we were able to prepare in high yield an ATPase species that only differs from intact ATPase because of excision of the MAATE 243 sequence from the loop linking the A domain with the third transmembrane segment. The PK-treated ATPase was unable to transport Ca 2؉ and to catalyze ATP hydrolysis, but it could bind two calcium ions with high affinity and react with ATP to form a classical ADPsensitive phosphoenzyme, Ca 2 E1P, with occluded Ca 2؉. The ability of Ca 2 E1P to become converted to the Ca 2؉free ADP-insensitive form, E2P, was strongly reduced, as was the ability of PK-treated ATPase to react with orthovanadate or to form an E2P intermediate from inorganic phosphate in the absence of Ca 2؉. PK-treated ATPase also reacted with thapsigargin to form a complex with altered properties, and the tryptic cleavage "T2" site in the A domain was no longer protected in the absence of Ca 2؉. It is probable that disrupting the C-terminal link of the A domain with the transmembrane region severely compromises reorientation of A and P domains and the functionally critical cross-talk of these domains with the membrane-bound Ca 2؉ ions.
Crystal structure of D351A mutant forms of the mammalian sarcoplasmic reticulum CA2+-ATPase reveals key events in phosphorylation and CA2+ release
HAL (Le Centre pour la Communication Scientifique Directe), 2008
... aide: Manuel et informations; Documentation utilisateur; Documentation WebServices; À propos.... more ... aide: Manuel et informations; Documentation utilisateur; Documentation WebServices; À propos. déposer. version française english version rss feed. ... A. Manchand, Am Winther, Pj Holm, C. Olesen, C. Montigny 1, 2 , B. Arnou 1, 2 , P. Champeil 1, 2 , Jd Clausen, B. Vilsen, Jp ...
Protein-protein contact contacts in solubilized membrane proteins, as detected by cross-linking
HAL (Le Centre pour la Communication Scientifique Directe), 2007
International audienc
Annual Review of Biophysics, 2011
Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep integ... more Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep integral membrane proteins (MPs) water soluble. In this review, we discuss their structure and solution behavior; the way they associate with MPs; and the structure, dynamics, and solution properties of the resulting complexes. All MPs tested to date form water-soluble complexes with APols, and their biochemical stability is in general greatly improved compared with MPs in detergent solutions. The functionality and ligand-binding properties of APol-trapped MPs are reviewed, and the mechanisms by which APols stabilize MPs are discussed. Applications of APols include MP folding and cell-free synthesis, structural studies by NMR, electron microscopy and X-ray diffraction, APol-mediated immobilization of MPs onto solid supports, proteomics, delivery of MPs to preexisting membranes, and vaccine formulation.
Inhibitors Bound to Ca<sup>2+</sup>-Free Sarcoplasmic Reticulum Ca<sup>2+</sup>−ATPase Lock Its Transmembrane Region but Not Necessarily Its Cytosolic Region, Revealing the Flexibility of the Loops Connecting Transmembrane and Cytosolic Domains
Biochemistry, Dec 1, 2007
Ca2+-free crystals of sarcoplasmic reticulum Ca2+-ATPase have, up until now, been obtained in the... more Ca2+-free crystals of sarcoplasmic reticulum Ca2+-ATPase have, up until now, been obtained in the presence of inhibitors such as thapsigargin (TG), bound to the transmembrane region of this protein. Here, we examined the consequences of such binding for the protein. We found that, after TG binding, an active site ligand such as beryllium fluoride can still bind to the ATPase and change the conformation or dynamics of the cytosolic domains (as revealed by the protection afforded against proteolysis), but it becomes unable to induce any change in the transmembrane domain (as revealed by the intrinsic fluorescence of the membranous tryptophan residues). TG also obliterates the Trp fluorescence changes normally induced by binding of MgATP or metal-free ATP, as well as those induced by binding of Mg2+ alone. In the nucleotide binding domain, the environment of Lys515 (as revealed by fluorescein isothiocyanate fluorescence after specific labeling of this residue) is significantly different in the ATPase complex with aluminum fluoride and in the ATPase complex with beryllium fluoride, and in the latter case it is modified by TG. All these facts document the flexibility of the loops connecting the transmembrane and cytosolic domains in the ATPase. In the absence of active site ligands, TG protects the ATPase from cleavage by proteinase K at Thr242-Glu243, suggesting TG-induced reduction in the mobility of these loops. 2,5-Di-tert-butyl-1,4-dihydroxybenzene or cyclopiazonic acid, inhibitors which also bind in or near the transmembrane region, also produce similar overall effects on Ca2+-free ATPase.
Unexpected Phosphoryl Transfer from Asp<sup>351</sup> to Fluorescein Attached to Lys515 in Sarcoplasmic Reticulum Ca<sup>2+</sup>-ATPase
Biochemistry, May 24, 2008
Sarcoplasmic reticulum Ca(2+)-ATPase is an ion pump whose catalytic cycle includes the transient ... more Sarcoplasmic reticulum Ca(2+)-ATPase is an ion pump whose catalytic cycle includes the transient formation of an acyl phosphate at Asp(351), and fluorescein isothiocyanate is a covalent inhibitor of ATP binding to this pump, known to specifically derivatize Lys(515) in the nucleotide-binding site. It was previously found that an unusually stable, phosphorylated form of fluorescein-ATPase, with low fluorescence, is obtained following Ca (2+) loading with acetyl phosphate as energy source and then chelation with EGTA of Ca(2+) on the cytosolic side. Here we show that the phospho-linkage in this low fluorescent species is stable at alkaline pH, unlike the acyl phosphate at Asp(351). Moreover, the low fluorescence and stable phosphoryl group track together in primary and secondary tryptic subfragments, separated by SDS-PAGE after denaturation. Finally, normal fluorescence and absorbance are recovered upon treatment with alkaline phosphatase after extensive trypsinolysis. We conclude that the low fluorescent species is the result of the phosphoryl group being transferred from Asp (351) to the fluorescein moiety during pump reversal, yielding fluorescein monophosphate tethered to Ca(2+)-ATPase.
Journal of Biological Chemistry, 1989
hus, Novo and the P.C. Petersen Foundation, by a short term fellowship from European Molecular Bi... more hus, Novo and the P.C. Petersen Foundation, by a short term fellowship from European Molecular Biology and Claude Bernard Association, and by a NATO traveling grant (to S. 0.
Biochemical and Biophysical Research Communications, 1973
The DNA conformational changes of B, A and C forms are reflected 5 in the infrared absorption spe... more The DNA conformational changes of B, A and C forms are reflected 5 in the infrared absorption spectra in the region of 800 cm-1 to 900 cm-1 and allow one to investigate unoriented samples. The transition to the A form is characterized by the appearence of bands at about 870 cm-1 and at 813 cm-l whereas the B and the C forms exhibit a band at 837 cm-l. these bands undoubtedly arise from phosphate diester stretching vibrations and yield information about backbone conformation. The presence of these infrared bands provides a criterion for testing the simultaneous presence of two coexisting forms of DNA. It represents a useful method for structural studies of nucleic acid complexes such as protein-DNA for which it is difficult to obtain orientation. Coprright @ 1973 by Academic Ress, Inc. A# rights of reproduction in any form reserved.
Ca2(+)-induced conformational changes and location of Ca2+ transport sites in sarcoplasmic reticulum Ca2(+)-ATPase as detected by the use of proteolytic enzyme (V8)
Journal of Biological Chemistry, 1990
Treatment of Ca2(+)-ATPase from sarcoplasmic reticulum with V8 protease from Staphylococcus aureu... more Treatment of Ca2(+)-ATPase from sarcoplasmic reticulum with V8 protease from Staphylococcus aureus produced appreciable amounts of a Ca2(+)-ATPase fragment (p85) in the presence of Ca2+ (E1 conformation of the enzyme), along with many other peptide fragments that were also formed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (E2 conformation). p85 was formed as a carboxyl-terminal cleavage product of Ca2(+)-ATPase by a split of the peptide bond between Glu-231 and Ile-232. Other conformation-dependent V8 splits were localized to the &quot;hinge&quot; region, involved in ATP binding, between the middle and COOH-terminal one-third of the Ca2(+)-ATPase polypeptide chain. Representative split products in this region (p48,p31) were identified as NH2-terminal and COOH-terminal cleavage products of p85. In the membrane p85 probably remains associated with its complementary NH2-terminal fragment(s) and retains the capacity to bind Ca2+ as evidenced by resistance to V8 degradation in Ca2+ and ability to become phosphorylated by ATP. However, the hydrolysis rate of the phosphorylated enzyme is reduced, indicating that peptide cleavage at Glu-231 interferes with Ca2+ transport steps after phosphorylation. Binding of Ca2+ to V8 and tryptic fragments of Ca2(+)-ATPase was studied on the basis of Ca2(+)-induced changes in electrophoretic mobility and 45Ca2+ autoradiography after transfer of peptides to Immobilon membranes. These data indicate binding by the NH2-terminal 1-198 amino acid residues (corresponding to the tryptic A2 fragment) and the COOH-terminal 715-1001 amino acid residues (corresponding to p31). By contrast the central portion of Ca2(+)-ATPase, including the NH2-terminal portion of p85, is devoid of Ca2+ binding. These results question an earlier proposition that Ca2(+)-binding is located to the &quot;stalk&quot; region of Ca2(+)-ATPase (Brandl, C. J., Green, N. M., Korczak, B., and MacLennan, D. H.) (1986) Cell 44, 597-607) but are in agreement with recent data obtained by oligonucleotide-directed mutagenesis of Ca2(+)-ATPase (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478). These different studies suggest that Ca2+ translocation sites may have an intramembranous location and are formed predominantly by the carboxyl-terminal part of the Ca2(+)-ATPase polypeptide chain.
Sarcoplasmic reticulum ATPase. Spin labeling detection of ligand-induced changes in the relative reactivities of certain sulfhydryl groups
Journal of Biological Chemistry, 1978
In sarcoplasmic reticulum fragments, chemical reactivity of calcium ATPase -SH groups toward N-(1... more In sarcoplasmic reticulum fragments, chemical reactivity of calcium ATPase -SH groups toward N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-iodoacetamide (ISL) was estimated by measuring the steady reduction in free label spectrum intensity during the labeling reaction. A few -SH groups reacted easily with ISL and activity was not inhibited. The reaction rate was highly sensitive to pH and temperature. Calcium chelation in the presence of magnesium accelerated the reaction slightly, and nucleotides accelerated if severalfold in the presence of calcium. The resulting spectra were also studied for the bound labels, after extensive washing of the nonreacted label. Compared to the spectrum obtained after labeling in the control calcium medium, the &quot;weakly immobilized signal&quot; of the spectrum of vesicles labeled in a chelated calcium medium was enhanced. On the other hand, the &quot;strongly immobilized signal&quot; was enhanced when vesicles were labeled in a medium containing calcium and nucleotides. This was taken as evidence that different -SH groups are selectively alkylated, according to the labeling medium. The present study confirms the calcium-induced modifications in the -SH environment reported previously and suggests new ways of searching for possible conformational events during the transport cycle in the membrane.
ATP regulation of sarcoplasmic reticulum Ca2+-ATPase. Metal-free ATP and 8-bromo-ATP bind with high affinity to the catalytic site of phosphorylated ATPase and accelerate dephosphorylation
Journal of Biological Chemistry, 1988
To localize and characterize the regulatory nucleotide site of skeletal muscle sarcoplasmic retic... more To localize and characterize the regulatory nucleotide site of skeletal muscle sarcoplasmic reticulum Ca2+-ATPase, we have investigated the effects of ADP, ATP, and analogues of these nucleotides on the rate of dephosphorylation of both native ATPase and ATPase modified with fluorescein 5&#39;-isothiocyanate (FITC), a reagent which hinders access of nucleotides to the ATPase catalytic site without affecting phosphorylation from Pi. Dephosphorylation of the phosphoenzyme formed from Pi was monitored by rapid filtration or stopped-flow fluorescence, mostly at 20 degrees C, pH 6.0, and in the absence of potassium. Fluorescence measurements were made possible through the use of 8-bromo-ATP, which selectively quenched certain tryptophan residues of the ATPase, thereby allowing the intrinsic fluorescence changes associated with dephosphorylation to be measured in the presence of bound nucleotide. ATP, 8-bromo-ATP, and trinitrophenyladenosine diand triphosphate, but not ADP, enhanced the rate of dephosphorylation of native ATPase 2-3-fold when added in the absence of divalent cations. Millimolar concentrations of Mg2+ eliminated the accelerating effects. Acceleration in the absence of Mg2+ was observed at relatively low concentrations of ATP and 8-bromo-ATP (0.01-0.1 mM) and binding of metal-free ATP and ADP, but not Mg.ATP, to the phosphoenzyme in this concentration range was demonstrated directly. Modification of the ATPase with FITC blocked nucleotide binding in the submillimolar concentration range and eliminated the nucleotide-induced acceleration of dephosphorylation. These results show that dephosphorylation, under these conditions, is regulated by ATP but not by Mg.ATP or ADP, and that the catalytic site is the locus of this &quot;regulatory&quot; ATP binding site.
Direct fluorescence measurements of Mg2+ binding to sarcoplasmic reticulum ATPase
Journal of Biological Chemistry, 1982
In the absence of calcium, interaction of magnesium with SR-ATPase induced a blue shift in intrin... more In the absence of calcium, interaction of magnesium with SR-ATPase induced a blue shift in intrinsic fluorescence emission. This Mg2+-induced fluorescence change was pH-dependent and an apparent Mg dissociation constant of 5 mM was found at pH 7. Equilibrium studies showed that magnesium competes for the high affinity Ca2+ binding sites and stopped flow measurements of the transient kinetics indicated a multistep interaction between magnesium and the calcium pump. These results suggest that magnesium drives the sarcoplasmic reticulum atpase toward an E.Mg species which might be a dead-end complex.
Rapid kinetics of myo-inositol trisphosphate binding and dissociation in cerebellar microsomes
Journal of Biological Chemistry, 1994
Using sheep cerebellum microsomes adsorbed on a filter, we measured the kinetics of [3H]inositol ... more Using sheep cerebellum microsomes adsorbed on a filter, we measured the kinetics of [3H]inositol 1,4,5-trisphosphate (InsP3) binding and dissociation on the subsecond time scale during rapid perfusion of the filter with [3H]InsP3-containing or InsP3-free media. At 20 degrees C and pH 7.1, in a cytosol-like medium containing MgCl2, the half-time for InsP3 dissociation was as short as 125 ms. The receptor behaved as a simple target for binding of its ligand, with the rate constant for InsP3 binding increasing linearly with InsP3 concentration. Various modulators of InsP3 binding (KCl, NaCl, pH, Mg2+, and Ca2+) were found to affect the receptor&#39;s apparent affinity for InsP3 mainly by altering the rate constant for [3H]InsP3 dissociation. ATP (but not InsP3) also accelerated [3H]InsP3 dissociation. In contrast to these modulators, luminal Ca2+ was found to have no effect on the amount of microsome-bound [3H]InsP3.
Kinetic characterization of the normal and detergent-perturbed reaction cycles of the sarcoplasmic reticulum calcium pump. Rate-limiting step(s) under different conditions
Journal of Biological Chemistry, 1986
We previously characterized the structural features of the interaction of sarcoplasmic reticulum ... more We previously characterized the structural features of the interaction of sarcoplasmic reticulum membranes with nonsolubilizing concentrations of C12E8, the non-ionic detergent octaethylene glycol monododecyl ether (Andersen, J.P., le Maire, M., Kragh-Hansen, V., Champeil, P., and Møller, J. V. (1983) Eur. J. Biochem. 134, 205-214). The present study characterizes especially the functional aspects and implications of the detergent-induced perturbation for an understanding of ATPase function. Perturbing detergent decreased Vmax, but left Ca2+ transport intact. Detergent incorporation affected neither the calcium-dependent phosphorylation from ATP, as judged from multimixer quenching experiments, nor the calcium-releasing transition between the two phosphoenzyme forms (Ca2E1P to E2P), as judged from kinetically resolved dual-wavelength measurements with the calcium-sensitive dye antipyrylazo III. However, the decrease in Vmax was accounted for by a decrease in the rate of enzyme dephosphorylation by a factor of 3-4, whereas the Ca2+-dependent transition between the nonphosphorylated enzyme forms (E2 to Ca2E1) was enhanced almost 10-fold. Evidence of a conformational change of E2 by C12E8 toward that of the E1 state to account for the perturbed reactions was obtained from experiments on vanadate reactivity and tryptic degradation pattern. Both direct and steady-state evidence was obtained for an acceleration by ATP of the Ca2E1P to E2P transition which may account for the low affinity modulatory effect of the nucleotide on enzyme turnover. The kinetic data indicated that reduction of ATP hydrolysis by C12E8 coincided with conditions where E2P dephosphorylation becomes rate-limiting (high ATP concentration, low pH, absence of potassium). Otherwise, the Ca2E1P to E2P transition is deduced to be a rate-limiting step for the ATPase cycle, whereas the potential for rate control of the cycle by modulation of the E2 to Ca2E1 transition is very small. Only in special circumstances (absence of potassium, high temperature, and using ITP as a substrate) did this transition become a rate-limiting step, subject to rate enhancement of the whole cycle by detergent perturbation.
Sarcoplasmic reticulum ATPase phosphorylation from inorganic phosphate in the absence of a calcium gradient. Steady state and kinetic fluorescence studies
Journal of Biological Chemistry, 1981
The intrinsic fluorescence of sarcoplasmic reticulum vesicles was measured under conditions allow... more The intrinsic fluorescence of sarcoplasmic reticulum vesicles was measured under conditions allowing ATPase phosphorylation from inorganic phosphate. Significant fluorescence enhancement of up to 4% resulted from gradient-independent enzyme phosphorylation at pH 6, in the absence of KCl. The equilibrium fluorescence data obtained at various magnesium and phosphate concentrations agree with a reaction scheme in which Mg2+, as direct activator, and free phosphate, as the true substrate, bind to the enzyme in random order to give a noncovalent ternary complex (Mg.*E.Pi), in equilibrium with the covalent phosphoenzyme (Mg.*E-P). The transient kinetics of the fluorescence rise was also studied, and the resulting data were generally consistent with the above scheme, assuming that binding reactions are fast compared to covalent phosphoenzyme formation. This, however, might be valid only as a first approximation. At 20 degrees C and pH 6, the phosphate concentration for half-maximum phosphorylation rate constant, at 20 mM magnesium, was higher than 20 mM. Similarly, the magnesium concentration for half-maximum phosphorylation rate constant, at 20 mM phosphate, was also higher than 20 mM. The maximum phosphorylation rate was faster than 25 s-1, and the phosphoenzyme hydrolysis rate constant was 1.5-2 s-1 under these conditions, so that the equilibrium constant between Mg.*E.Pi and Mg.*E-P largely favors the phosphoenzyme.
A direct fluorescence study of the transient steps induced by calcium binding to sarcoplasmic reticulum ATPase
Journal of Biological Chemistry, 1980
The sarcoplasmic reticulum intrinsic fluorescence level was closely correlated with the ATPase fu... more The sarcoplasmic reticulum intrinsic fluorescence level was closely correlated with the ATPase functional state, from pH 5.5 to 8.5. The fluorescence signal was used in stopped flow measurements for direct study of transient pump kinetics after calcium binding or removal. The signal change time course, which depends solely on the free calcium concentration in the observation chamber, was analyzed as a single exponential. Rate constants (kobs) were relatively slow (5 to 20 s-1), indicating multistep interaction between calcium and the transport protein. At pH 7 and 20 degrees C, and in the presence of 100 mM potassium and 1 to 20 mM MgCl2, kobs first decreased, and then increased as the calcium concentration rose. Similar experiments were performed at pH 6. Data were analyzed according to a scheme in which sarcoplasmic reticulum . calcium complex formation is controlled by a slow isomerization step occurring either before or after the rapid calcium binding to the high affinity site. The results are discussed with reference to published rapid quenching experiments. Under our conditions, i.e. in the absence of a calcium gradient across the membrane, the calcium pump cycle step in which reorientation of the calcium binding sites occurs cannot be identified with the isomerization step mentioned above.
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Papers by Philippe Champeil