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J J Lacapère

Publications and source records attributed to J J Lacapère.

At least 19 recordsLinked to original sources
PubMed2001

Structural and functional study of reconstituted peripheral benzodiazepine receptor.

Recombinant mouse 18 kDa peripheral-type benzodiazepine receptor (PBR) protein was overexpressed in Escherichia coli and isolated using a His. Bind metal chelation resin. Recombinant PBR protein was purified with sodium dodecyl sulfate and reincorporated into liposomes using Bio-Beads SM2 as a detergent removing agent. Negative staining of the reconstituted PBR samples, examined by electron microscopy, showed the formation of proteoliposomes. Freeze-fracture of these proteoliposomes revealed the presence of transmembranous particles of an average size of 3.5 +/- 0.25 nm, consistent with the presence of a monomeric form of the recombinant PBR protein. The reconstituted protein exhibited the ability to bind both the PBR drug ligand isoquinoline carboxamide PK 11195 and cholesterol with nanomolar affinities. These data suggest that a PBR monomer is the minimal functional unit, binding drug ligands and cholesterol.

Animals↗
PubMed2001

Phosphorylated Ca2+-ATPase stable enough for structural studies.

The atomic structure of sarcoplasmic reticulum Ca(2+)-ATPase, in a Ca(2+)-bound conformation, has recently been elucidated (Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. (2000) Nature 405, 647-655). Important steps for further understanding the mechanism of ion pumps will be the atomic structural characterization of different key conformational intermediates of the transport cycle, including phosphorylated intermediates. Following our previous report (Champeil, P., Henao, F., Lacapère, J.-J. & McIntosh, D. B. (2000) J. Biol. Chem. 276, 5795-5803), we show here that it is possible to prepare a phosphorylated form of sarcoplasmic reticulum Ca(2+)-ATPase (labeled with fluorescein isothiocyanate) with a week-long stability both in membranes and in mixed lipid-detergent micelles. We show that this phosphorylated fluorescein isothiocyanate-ATPase can form two-dimensional arrays in membranes, similar to those that were used previously to reconstruct from cryoelectron microscopy images the three-dimensional structure of Ca(2+)-free unphosphorylated ATPase. The results also provide hope that crystals of phosphorylated Ca(2+)-ATPase suitable for x-ray crystallography will be achieved.

Animals↗
PubMed2000

Efficient solubilization and purification of the gastric H+, K+-ATPase for functional and structural studies.

When gastric H(+),K(+)-ATPase-containing microsomes are solubilized by detergents, a rapid loss of ATPase activity is generally observed. In this article, SDS/PAGE of octa(ethylene glycol)dodecyl monoether (C(12)E(8))- and n-dodecyl beta-d-maltoside-solubilized microsomes and their purifications by affinity chromatography on Reactive Red column reveal that inactivation is due to two main effects. (i) Solubilization activates an aspartic protease that cleaves down the alpha-subunit of the H(+),K(+)-ATPase. Addition of pepstatin A at slightly acidic pH and at low temperature prevents the proteolysis. (ii) A too-harsh delipidation inactivates the ATPase. When n-dodecyl-beta-d-maltoside is the detergent, the soluble H(+), K(+)-ATPase is highly active (2.5 micromol/mg per h at pH 6.0 and 5 degrees C) as long as ATP is added. When C(12)E(8) is used, the detergent induces an inactivation due to delipidation, since addition of lipids restores activity. The two subunits of the H(+), K(+)-ATPase are present in equimolar ratio in the n-dodecyl beta-d-maltoside-purified complex. Moreover, two main types of complex (330 and 660 kDa) were resolved in non-denaturing gels and should be the dimeric (alphabeta)(2) and tetrameric (alphabeta)(4) heterodimers respectively. In conclusion, purification of active, stable, soluble complexes of H(+),K(+)-ATPase with few lipids (a lipid/protein ratio of 0.25, w/w) has been achieved. This material should be useful for further structural studies.

Adenosine Triphosphate↗
PubMed1999

The 9 A projection structure of cytochrome b6f complex determined by electron crystallography.

Thin three-dimensional crystals of the cytochrome b6 f complex from the unicellular algae Chlamydomonas reinhardtii have been grown by BioBeads-mediated detergent removal from a mixture of protein and lipid solubilized in Hecameg. Frozen-hydrated crystals, exhibiting p22121 plane group symmetry, were studied by electron crystallography and a projection map at 9 A resolution was calculated. The crystals (unit cell dimensions of a=173.5 A, b=70.0 A and gamma=90.0 degrees) showed the presence of dimers, and within each monomer 14 domains of electron density were observed. The combination of the projection map obtained from ice-embedded crystals of cytochrome b6 f with a previous map obtained from negatively stained samples brings new insight in the organization of the complex. For example, it distinguishes some peaks and/or domains that are only extramembrane or transmembrane, and reveals the possible localization of single-stranded transmembrane alpha-helices (Pet subunits). Furthermore, the cross-correlation of our projection map from frozen hydrated samples with the atomic model of the transmembrane part of the cytochrome bc1 complex has allowed us to localize the cytochrome b6 at the dimer interface and to reveal structural differences between the two complexes.

Animals↗
PubMed1998

A hexameric transmembrane pore revealed by two-dimensional crystallization of the large mechanosensitive ion channel (MscL) of Escherichia coli.

We have established a reconstitution method of the detergent-solubilized recombinant large mechanosensitive ion channel of Escherichia coli (MscL) that yielded two-dimensional crystals. For that purpose, we have developed a new protocol using Triton X-100 to solubilize and purify the MscL protein. This protocol not only allowed an increase in the protein yield but also made it possible to obtain a homogeneous delipidated and reproducible preparation of the purified protein. When examined by the patch-clamp method MscL channels were found to be fully functional, exhibiting characteristic conductance and activation by pressure. For electron crystallography the homogeneous Triton X-100-purified recombinant MscL was further reconstituted at low lipid-to-protein ratios using Bio-Beads SM2 to remove the detergent. Two-dimensional crystals, exhibiting a p6 plane group symmetry, have been produced and examined by negative stain electron microscopy. Image processing of selected micrographs yielded a projection map at 15-A resolution that provided the first explicit structural information about the molecular boundary and homohexameric organization of the MscL channels in the membrane bilayer.

Bacterial Proteins↗
PubMed1998

Interaction of a Golgi-associated kinesin-like protein with Rab6.

Rab guanosine triphosphatases regulate vesicular transport and membrane traffic within eukaryotic cells. Here, a kinesin-like protein that interacts with guanosine triphosphate (GTP)-bound forms of Rab6 was identified. This protein, termed Rabkinesin-6, was localized to the Golgi apparatus and shown to play a role in the dynamics of this organelle. The carboxyl-terminal domain of Rabkinesin-6, which contains the Rab6-interacting domain, inhibited the effects of Rab6-GTP on intracellular transport. Thus, a molecular motor is a potential effector of a Rab protein, and coordinated action between members of these two families of proteins could control membrane dynamics and directional vesicular traffic.

Adenosine Triphosphatases↗
PubMed1998

Acting on actin: the electric motility assay.

We have developed a novel technique which allows one to direct the two dimensional motion of actin filaments on a myosin coated sheet using a weak electric field parallel to the plane of motion. The filament velocity can be increased or decreased, and even reversed, as a function of orientation and strength of the field. PMMA (poly(methylmethacrylate)) gratings, which act as rails for actin, allow one for the first time to explore three quadrants of the force velocity diagram. We discuss effective friction, duty ratio and stall force at different myosin densities. A discontinuity in the velocity force relationship suggests the existence of dynamical phase transition.

Actins↗
PubMed1998

Two-dimensional crystallization of Ca-ATPase by detergent removal.

By using Bio-Beads as a detergent-removing agent, it has been possible to produce detergent-depleted two-dimensional crystals of purified Ca-ATPase. The crystallinity and morphology of these different crystals were analyzed by electron microscopy under different experimental conditions. A lipid-to-protein ratio below 0.4 w/w was required for crystal formation. The rate of detergent removal critically affected crystal morphology, and large multilamellar crystalline sheets or wide unilamellar tubes were generated upon slow or fast detergent removal, respectively. Electron crystallographic analysis indicated unit cell parameters of a = 159 A, b = 54 A, and gamma = 90 degrees for both types of crystals, and projection maps at 15-A resolution were consistent with Ca-ATPase molecules alternately facing the two sides of the membrane. Crystal formation was also affected by the protein conformation. Indeed, tubular and multilamellar crystals both required the presence of Ca2+; the presence of ADP gave rise to another type of packing within the unit cell (a = 86 A, b = 77 A, and gamma = 90 degrees), while maintaining a bipolar orientation of the molecules within the bilayer. All of the results are discussed in terms of nucleation and crystal growth, and a model of crystallogenesis is proposed that may be generally true for asymmetrical proteins with a large hydrophilic cytoplasmic domain.

Animals↗
PubMed1997

How to make tubular crystals by reconstitution of detergent-solubilized Ca2(+)-ATPase.

In an attempt to better define the parameters governing reconstitution and two-dimensional crystallization of membrane proteins, we have studied Ca2(+)-ATPase from rabbit sarcoplasmic reticulum. This ion pump forms vanadate-induced crystals in its native membrane and has previously been reconstituted at high lipid-to-protein ratios for functional studies. We have characterized the reconstitution of purified Ca2(+)-ATPase at low lipid-to-protein ratios and discovered procedures that produce long, tubular crystals suitable for helical reconstruction. C12E8 (n-dodecyl-octaethylene-glycol monoether) was used to fully solubilize various mixtures of lipid and purified Ca2(+)-ATPase, and BioBeads were then used to remove the C12E8. Slow removal resulted in two populations of vesicles, and the proteoliposome population was separated from the liposome population on a sucrose density gradient. These proteoliposomes had a lipid-to-protein ratio of 1:2, and virtually 100% of molecules faced the outside of vesicles, as determined by fluorescein isothiocyanate labeling. Cycles of freeze-thaw caused considerable aggregation of these proteoliposomes, and, if phosphatidyl ethanolamine and phosphatidic acid were included, or if the bilayers were doped with small amounts of C12E8, vanadate-induced tubular crystals grew from the aggregates. Thus our procedure comprised two steps-reconstitution followed by crystallization-allowing us to consider mechanisms of bilayer formation separately from those of crystallization and tube formation.

Animals↗
PubMed1996

Manganese as a cosubstrate for the phosphorylation of the sarcoplasmic reticulum Ca-dependent adenosine triphosphatase with orthophosphate.

The phosphorylation of the sarcoplasmic reticulum Ca-ATPase (EC 3.6.1.38) with P(i) was characterized using Mn as a Mg analogue. Steady state and transient fluorescence and radioisotopic techniques were used; the affinities of Mn and P(i) for the enzyme and the rate constants of the phosphorylation and dephosphorylation reactions were determined, under several conditions. The reactions were carried out at pH 5.5 to minimize the binding of contaminant Ca to the transport sites, thus avoiding the use of Ca chelators. The apparent affinity of Mn binding at low [Mn] is larger in the absence of P(i) (35 microM) than in the presence of saturating P(i) (70 microM). On the contrary, the apparent affinity of Mn for the formation of the phosphoenzyme increases, from 1.5 mM to 0.15 mM, upon increasing [P(i)] in the millimolar range. The apparent affinty of P(i) for the formation of the phosphoenzyme also increases, from 2.2 mM to 0.2 mM, upon increasing [Mn] in the millimolar range. The equilibrium of the phosphoenzyme with the noncovalent Mn.P(i). Enzyme complex favors the covalent species. The simulation of a reaction model including the random binding of 2 Mn and I P(i) per mol of ATPase and a noncovalent complex in equilibrium with the phosphoenzyme, using a set of equilibrium constants deduced from the results, agree with the experimental data.

Animals↗
PubMed1994

Conformation of Ca(2+)-ATPase in two crystal forms. Effects of Ca2+, thapsigargin, adenosine 5'-(beta, gamma-methylene)triphosphate), and chromium(III)-ATP on crystallization.

The structure of Ca(2+)-ATPase has been studied by electron microscopy of two different crystal forms: one tubular form induced by vanadate in native sarcoplasmic reticulum (SR) membranes and another multilamellar form grown from detergent-solubilized SR. To determine the conformation of Ca(2+)-ATPase within each crystal form, the respective effects of Ca2+, thapsigargin, adenosine 5'-(beta, gamma-methylene)triphosphate) (AMP-PCP), and chromium(III) (Cr-ATP) on crystallization have been studied. Vanadate-induced tubes were prevented from forming by micromolar Ca2+, but if preformed in the absence of Ca2_, millimolar Ca2+ was required to disrupt these crystals. Thapsigargin promoted tube formation even in the presence of 10 mM Ca2+. Neither AMP-PCP nor Cr-ATP prevented tube formation, and the Ca2+ sensitivity of tube formation from Cr-ATP-inhibited SR was identical to controls. Multilamellar crystals required at least 0.2 mM Ca2+ and were prevented from forming by thapsigargin, AMP-PCP, or Cr-ATP. It is concluded that helical tubes are composed of the Ca(2+)-free, dephosphorylated conformation (E), and the nucleotide-bound conformation (E-ATP) is also tolerated. In contrast, multilamellar crystals are composed of the Ca(2+)-bound conformation (E.Ca2) and do not tolerate nucleotide binding. Thus, comparison of structures obtained from the two crystal forms should reveal physiologically relevant conformational differences.

Adenosine Triphosphate↗
PubMed1994

Interaction of 4-azido-2-nitrophenyl phosphate, a pseudosubstrate, with the sarcoplasmic reticulum Ca-ATPase.

In the dark, 4-azido-2-nitrophenyl phosphate (ANPP) is a phosphate analog which behaves like a simple energy-rich phosphate donor for the sarcoplasmic reticulum (SR) Ca-ATPase. Like p-nitrophenyl phosphate (pNPP), ANPP is hydrolyzed by the enzyme only in the presence of calcium and magnesium (K0.5 and Vmax are 0.3 mM and 60 nmol mg-1 min-1, respectively). After photoirradiation in the absence of magnesium, SR Ca-ATPase is specifically labeled by [32P]ANPP in the presence and in the absence of calcium. The presence of nucleotide in the medium provides some protection against photolabeling but less than phosphorylation by inorganic phosphate. The maximal stoichiometry of covalently bound ANPP extrapolates to 0.8 mol/mol of Ca-ATPase in the absence of magnesium. Autoradiography of a sodium dodecyl sulfate-polyacrylamide gel, after controlled trypsin digestion of the photolabeled protein, reveals that the radioactivity is incorporated in the B subfragment. Three radioactive polypeptides with approximate molecular masses of 55, 25, and 6 kDa are obtained depending on the digestion conditions. N-Terminal sequence analysis of the 50- and 25-kDa peptides reveals the same sequence beginning at Ala-506, whereas two different sequence beginning at Ala-506 and Phe-584 are observed in the 6-kDa peptides.

Adenosine Triphosphate↗
PubMed1993

Identification of amino acid residues photolabeled with 8-azidoadenosine 5'-diphosphate in the catalytic site of sarcoplasmic reticulum Ca-ATPase.

The photoreactive ADP analogue 8-N3-ADP binds in the dark to the catalytic site of the sarcoplasmic reticulum Ca-ATPase. An apparent Kd value of 30 microM has been deduced from competition with ADP in the presence of EGTA. Photoirradiation of Ca-ATPase with 8-N3-[3H]ADP in the presence of calcium results in irreversible inhibition of ATPase activity with corresponding stoichiometries of covalently and specifically photolabeled Ca-ATPase. The site of photolabeling of the Ca-ATPase in the presence of calcium has been explored. Controlled trypsin digestion of the labeled protein shows that 8-azido-ADP is incorporated in the B subfragment. Extensive trypsin digestion of the labeled protein releases a small peptide as revealed by gel filtration chromatography (Sephadex G-50). Further HPLC purification on a reverse-phase column (C8) eluted with a water/acetonitrile gradient buffered at pH 6 or at pH 2 gives a single labeled peptide. Edman degradation of that isolated peptide, as well as the amino acid composition, shows that it contains five amino acid residues (Val-530-Arg-534) with the radioactivity localized on Thr-532 and Thr-533.

Adenosine Diphosphate↗
PubMed1992

Atomic force microscopy of three-dimensional membrane protein crystals. Ca-ATPase of sarcoplasmic reticulum.

We have observed three-dimensional crystals of the calcium pump from sarcoplasmic reticulum by atomic force microscopy (AFM). From AFM images of dried crystals, both on graphite and mica, we measured steps in the crystal thickness, corresponding to the unit cell spacing normal to the substrate. It is known from transmission electron microscopy that crystal periodicity in the plane of the substrate is destroyed by drying, and it was therefore not surprising that we were unable to observe this periodicity by AFM. Thus, we were motivated to use the AFM on hydrated crystals. In this case, crystal adsorption appeared to be a limiting factor, and our studies indicate that adsorption is controlled by the composition of the medium and by the physical-chemical properties of the substrate. We used scanning electron microscopy to determine the conditions yielding the highest adsorption of crystals, and, under these conditions, we have obtained AFM images of hydrated crystals with a resolution similar to that observed with dried samples (i.e., relatively poor). In the same preparations, we have observed lipid bilayers with a significantly better resolution, indicating that the poor quality of crystal images was not due to instrumental limitations. Rather, we attribute poor images to the intrinsic flexibility of these multilamellar crystals, which apparently allow movement of one layer relative to another in response to shear forces from the AFM tip. We therefore suggest some general guidelines for future studies of membrane proteins with AFM.

Animals↗
PubMed1990

Reversal of the sarcoplasmic reticulum ATPase cycle by substituting various cations for magnesium. Phosphorylation and ATP synthesis when Ca2+ replaces Mg2+.

Reversal of the cycle of sarcoplasmic reticulum ATPase starts from ATPase phosphorylation by Pi, in the presence of Mg2+, and leads to ATP synthesis. We show here that ATP can also be synthesized when Ca2+ replaces Mg2+. In the absence of a calcium gradient and in the presence of dimethyl sulfoxide, ATPase phosphorylation from Pi and Ca2+ led to the formation of an unstable phosphoenzyme. This instability was due to a competition between the phosphorylation reaction induced by Pi and Ca2+ and the transition induced by Ca2+ binding to the transport sites, which led to a conformation that could not be phosphorylated from Pi. Dimethyl sulfoxide and low temperature stabilized the calcium phosphoenzyme, which under appropriate conditions, subsequently reacted with ADP to synthesize ATP. Substitution of Co2+, Mn2+, Cd2+, or Ni2+ for Mg2+ induced ATPase phosphorylation from Pi, giving phosphoenzymes of various stabilities. However, substitution of Ba2+, Sr2+, or Cr3+ produced no detectable phosphoenzymes, under the same experimental conditions. Our results show that ATPase phosphorylation from Pi, like its phosphorylation from ATP, does not have a strict specificity for magnesium.

Adenosine Triphosphatases↗
PubMed1990

pH and magnesium dependence of ATP binding to sarcoplasmic reticulum ATPase. Evidence that the catalytic ATP-binding site consists of two domains.

Nucleotide binding to sarcoplasmic reticulum vesicles was investigated in the absence of calcium using both filtration and fluorescence measurements. Filtration assays of binding of radioactive nucleotides at concentrations up to 0.1 mM gave a stoichiometry of one ATP-binding site/sarcoplasmic reticulum ATPase molecule. When measured in the presence of calcium under otherwise similar conditions, ATPase velocity rose 4-8-fold (depending on pH and magnesium concentration) when the ATP concentration was increased from 1 microM to 0.1 mM. Binding of ATP and ADP enhanced the intrinsic fluorescence of sarcoplasmic reticulum ATPase, but AMP and adenosine did not affect it. Both filtration and fluorescence measurements showed that binding of metal-free ATP is independent of pH (Kd = 20-25 microM) but that the presence of magnesium induces pH dependence of the binding of the Mg.ATP complex (Kd = 10 microM at pH 6.0 and 1.5 microM at pH 8.0). Binding of metal-free ADP was pH-dependent but was not affected by magnesium. High magnesium concentrations inhibited nucleotide binding. These results suggest that ATP interacts with two different domains of Ca-ATPase that form the catalytic site. The first domain may bind the adenine moiety of the substrate, and the pH dependence of ADP binding suggests the participation of His683 in this region. The second domain of the catalytic site may bind the gamma-phosphate and the magnesium ion of the Mg.ATP complex and constitute the locus of the electrostatic interactions between the substrate and the enzyme.

Adenosine Diphosphate↗