Biomedical subjects

Jean-Luc Popot

Publications and source records attributed to Jean-Luc Popot.

18 recordsLinked to original sources

PubMed2007

Fluorinated and hemifluorinated surfactants as alternatives to detergents for membrane protein cell-free synthesis.

Hemifluorinated and fluorinated surfactants are lipophobic and, as such, non-detergent. Although they do not solubilize biological membranes, they can, after conventional solubilization, substitute for detergents to keep membrane proteins soluble, which generally improves their stability [Breyton, Chabaud, Chaudier, Pucci and Popot (2004) FEBS Lett. 564, 312-318]. In the present study, we show that (hemi)fluorinated surfactants can be used for in vitro synthesis of membrane proteins: they do not interfere with protein synthesis, and they provide a suitable environment for MscL, a pentameric mechanosensitive channel, to fold and oligomerize to its native functional state. Following synthesis, both types of surfactants can be used to deliver MscL directly to pre-formed lipid vesicles. The electrophysiological activity of MscL synthesized in vitro in the presence of either hemi- or per-fluorinated surfactant is similar to that of the protein expressed in vivo.

Cell Membrane↗

PubMed2006

Amphipathic polymers: tools to fold integral membrane proteins to their active form.

Among the major obstacles to pharmacological and structural studies of integral membrane proteins (MPs) are their natural scarcity and the difficulty in overproducing them in their native form. MPs can be overexpressed in the non-native state as inclusion bodies, but inducing them to achieve their functional three-dimensional structure has proven to be a major challenge. We describe here the use of an amphipathic polymer, amphipol A8-35, as a novel environment that allows both beta-barrel and alpha-helical MPs to fold to their native state, in the absence of detergents or lipids. Amphipols, which are extremely mild surfactants, appear to favor the formation of native intramolecular protein-protein interactions over intermolecular or protein-surfactant ones. The feasibility of the approach is demonstrated using as models OmpA and FomA, two outer membrane proteins from the eubacteria Escherichia coli and Fusobacterium nucleatum, respectively, and bacteriorhodopsin, a light-driven proton pump from the plasma membrane of the archaebacterium Halobacterium salinarium.

Bacterial Outer Membrane Proteins↗

PubMed2006

Lactobionamide surfactants with hydrogenated, perfluorinated or hemifluorinated tails: physical-chemical and biochemical characterization.

Detergents are customarily used to solubilize cell membranes and keep membrane proteins soluble in aqueous buffers, but they often lead to irreversible protein inactivation. Hemifluorinated amphiphiles with hybrid hydrophobic chains have been specifically designed to minimize the denaturating propensity of surfactants toward membrane proteins. We have studied the physical-chemical and biochemical properties of lactobionamide surfactants bearing either a hydrogenated, a fluorinated or a hemifluorinated chain (respectively H-, F-, and HF-Lac). We show that the dual composition of the hydrophobic chain of HF-Lac endows it with unusual physical-chemical properties as regards its critical micellar concentration, interfacial area per molecule, and behavior upon reverse phase chromatography. Analytical ultracentrifugation shows that, whereas H-Lac assembles into well-defined micelles, F-Lac and HF-Lac form large and heterogeneous assemblies, whose size increases with surfactant concentration. Molecular dynamics calculations suggest that F-Lac forms cylindrical micelles. The ability of HF-Lac to keep membrane proteins soluble was examined using the cytochrome b(6) f complex from Chlamydomonas reinhardtii's chloroplast as a model protein. HF-Lac/b(6) f complexes form particles relatively homogeneous in size, in which the b(6) f complex is as stable or markedly more stable, depending on the surfactant concentration, than it is in equivalent concentrations of hydrogenated surfactants, including H-Lac.

Biochemical Phenomena↗

PubMed2006

Fluorinated and hemifluorinated surfactants derived from maltose: synthesis and application to handling membrane proteins in aqueous solution.

Two novel fluorinated surfactants have been obtained by grafting by radical reaction either a fluorocarbon or an ethyl end-capped fluorocarbon chain onto the double bond of beta-D-allyl maltose. The two compounds thus obtained form polydisperse aggregates in water. They can keep membrane proteins water-soluble, but the protein/surfactant complexes are polydisperse, which affects neither the native state nor the stability of the proteins.

Hydrocarbons, Fluorinated↗

PubMed2006

Chaperoning of insertion of membrane proteins into lipid bilayers by hemifluorinated surfactants: application to diphtheria toxin.

Hemifluorinated compounds, such as HF-TAC, make up a novel class of nondetergent surfactants designed to keep membrane proteins soluble under nondissociating conditions [Breyton, C., et al. (2004) FEBS Lett. 564, 312]. Because fluorinated and hydrogenated chains do not mix well, supramicellar concentrations of these surfactants can coexist with intact lipid vesicles. To test the ability of HF-TAC to assist proper membrane insertion of proteins, we examined its effect on the pH-triggered insertion of the diphtheria toxin T-domain. The function of the T-domain is to translocate the catalytic domain across the lipid bilayer in response to acidification of the endosome. This translocation is accompanied by the formation of a pore, which we used as a measure of activity in a vesicle leakage assay. We have also used Förster resonance energy transfer to follow the effect of HF-TAC on aggregation of aqueous and membrane-bound T-domain. Our data indicate that the pore-forming activity of the T-domain is affected by the dynamic interplay of two principal processes: productive pH-triggered membrane insertion and nonproductive aggregation of the aqueous T-domain at low pH. The presence of HF-TAC in the buffer is demonstrated to suppress aggregation in solution and ensure correct insertion and folding of the T-domain into the lipid vesicles, without solubilizing the latter. Thus, hemifluorinated surfactants stabilize the low-pH conformation of the T-domain as a water-soluble monomer while acting as low-molecular weight chaperones for its insertion into preformed lipid bilayers.

Diphtheria Toxin↗

PubMed2006

Protective and inhibitory effects of various types of amphipols on the Ca2+-ATPase from sarcoplasmic reticulum: a comparative study.

Amphipols are amphipathic polymers designed to replace or supplement detergents in membrane protein solution studies. Previous work has suggested both advantages and disadvantages to the use of a polyacrylate-based amphipol, A8-35, for studying the sarcoplasmic reticulum Ca2+-ATPase (SERCA1a). We investigated this issue further using a set of four amphipols with different chemical structures. Previous size exclusion chromatography experiments had shown that A8-35 and SERCA1a/A8-35 complexes aggregate under certain conditions. We show here that aggregation can be prevented by omitting calcium from buffers or by using a sulfonated version of A8-35. A8-35 had previously been shown to protect Ca2+-ATPase from irreversible denaturation, while inhibiting its activity in a reversible manner. We show here that the other three amphipols tested also display these properties and that all four amphipols slow down backward calcium dissociation from the nonphosphorylated solubilized enzyme, a priori an unrelated step. As this calcium dissociation involves the opening up of the bundle of transmembrane ATPase segments, the slowing of this process may indicate that multipoint attachment of the polymers to the hydrophobic transmembrane surface damps protein dynamics ("Gulliver" effect). Damping might be the reason why amphipols also simultaneously protect membrane proteins against irreversible denaturation and may inhibit the activity of those of them that display large rearrangements of their transmembrane surface during their catalytic cycle.

Animals↗

PubMed2006

Well-defined nanoparticles formed by hydrophobic assembly of a short and polydisperse random terpolymer, amphipol A8-35.

Amphipols are short amphilic polymers designed for applications in membrane biochemistry and biophysics and used, in particular, to stabilize membrane proteins in aqueous solutions. Amphipol A8-35 was obtained by modification of a short-chain parent polymer (poly(acrylic acid); PAA) with octyl- and isopropylamine, to yield an amphiphilic product with an average molar mass of 9-10 kg x mol(-1) (sodium salt form) and a polydispersity index of 2.0 to 3.1, depending on the source of PAA. The behavior of A8-35 in aqueous buffers was studied by size exclusion chromatography, static and dynamic light scattering, equilibrium and sedimentation velocity analytical ultracentrifugation, and small angle neutron scattering. Despite the variable length of the chains and the random distribution of hydrophobic groups along them, A8-35 self-organizes into well-defined assemblies. The data are best compatible with most of the polymer forming compact assemblies (particles) with a molar mass of approximately 40 kg x mol(-1), a radius of gyration of approximately 2.4 nm, and a Stokes radius of approximately 3.15 nm. Each particle contains, on average, four A8-35 macromolecules and 75-80 octyl chains. Neutron scattering reveals a sharp interface between the particles and water. A minor (approximately 0.1%) mass fraction of the material forms much larger aggregates, whose proportion may increase under certain conditions of preparation or handling, such as low pH. They can be removed by gel filtration.

Chromatography, Gel↗

PubMed2005

Reconstitution and alignment by a magnetic field of a beta-barrel membrane protein in bicelles.

A protocol is described for the reconstitution of a transmembrane beta-barrel protein domain, tOmpA, into lipid bicelles. tOmpA is the largest protein to be reconstituted in bicelles to date. Its insertion does not prevent bicelles from orienting with their plane either parallel or perpendicular to the magnetic field, depending on the absence or presence of paramagnetic ions. In the latter case, tOmpA is shown to align with the axis of the beta-barrel parallel to the magnetic field, i.e. perpendicular to the plane of the bilayer, an orientation conforming to that in natural membranes and favourable to structural studies by solid-state NMR. Reconstitution into bicelles may offer an interesting approach for structural studies of membrane proteins in a medium resembling a biological membrane, using either NMR or other biophysical techniques. Our data suggest that alignment in the magnetic field of membrane proteins included into bicelles may be facilitated if the protein is folded as a beta-barrel structure.

Analytic Sample Preparation Methods↗

PubMed2005

NMR study of a membrane protein in detergent-free aqueous solution.

One of the major obstacles to membrane protein (MP) structural studies is the destabilizing effect of detergents. Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep MPs water-soluble under mild conditions. In the present work, we have explored the feasibility of studying the structure of APol-complexed MPs by NMR. As a test MP, we chose the 171-residue transmembrane domain of outer MP A from Escherichia coli (tOmpA), whose x-ray and NMR structures in detergent are known. 2H,15N-labeled tOmpA was produced as inclusion bodies, refolded in detergent solution, trapped with APol A8-35, and the detergent removed by adsorption onto polystyrene beads. The resolution of transverse relaxation-optimized spectroscopy-heteronuclear single-quantum correlation spectra of tOmpA/A8-35 complexes was found to be close to that of the best spectra obtained in detergent solutions. The dispersion of chemical shifts indicated that the protein had regained its native fold and retained it during the exchange of surfactants. MP-APol interactions were mapped by substituting hydrogenated for deuterated A8-35. The resulting dipolar broadening of amide proton linewidths was found to be limited to the beta-barrel region of tOmpA, indicating that A8-35 binds specifically to the hydrophobic transmembrane surface of the protein. The potential of this approach to MP studies by solution NMR is discussed.

Amino Acid Sequence↗

PubMed2004

Partial specific volume and solvent interactions of amphipol A8-35.

Amphipols are small amphiphilic polymers that can stabilize and keep soluble membrane proteins in aqueous solutions in the absence of detergent. A prerequisite to solution studies of membrane protein/amphipol complexes is the determination of the partial specific volume v2 and effective charge z of the polymer. The ratio (R) of the buoyant molar masses of particles in D2O and H2O solutions, obtained from sedimentation velocity (sH/sD method) and sedimentation equilibrium experiments, and their contrast match point (CMP), determined in small-angle neutron scattering experiments, depend on v2 and z. When z is known, v2 can be estimated from R with a good accuracy as long as v2 is close to 1. The effects of labile H/D exchange and of polyelectrolyte counter-ion dissociation in general cannot be neglected. The accuracy, advantages, and limits of the sH/sD method have been studied in details using model macromolecules (DNA, protein, and polysaccharide). The sH/sD method appears particularly advantageous for the study of heterogeneous samples. Measurements of density, sD/sH buoyant molar masses in H2O, D2O, and D2(18)O, and CMP of hydrogenated and partially deuterated A8-35, a polyacrylate-based amphipol containing 35 underivatized carboxylates per 100 monomers, led to a consistent description of its buoyancy and charge properties.

Carbon↗

PubMed2004

Hemifluorinated surfactants: a non-dissociating environment for handling membrane proteins in aqueous solutions?

The instability of membrane proteins in detergent solution can generally be traced to the dissociating character of detergents and often correlates with delipidation. We examine here the possibility of substituting detergents, after membrane proteins have been solubilized, with non-detergent surfactants whose hydrophobic moiety contains a perfluorinated region that makes it lipophobic. In order to improve its affinity for the protein surface, the fluorinated chain is terminated by an ethyl group. Test proteins included bacteriorhodopsin, the cytochrome b(6)f complex, and the transmembrane region of the bacterial outer membrane protein OmpA. All three proteins were purified using classical detergents and transferred into solutions of C(2)H(5)C(6)F(12)C(2)H(4)-S-poly-Tris-(hydroxymethyl)aminomethane (HF-TAC). Transfer to HF-TAC maintained the native state of the proteins and prevented their precipitation. Provided the concentration of HF-TAC was high enough, HF-TAC/membrane protein complexes ran as single bands upon centrifugation in sucrose gradients. Bacteriorhodopsin and the cytochrome b(6)f complex, both of which are detergent-sensitive, exhibited increased biochemical stability upon extended storage in the presence of a high concentration of HF-TAC as compared to detergent micelles. The stabilization of cytochrome b(6)f is at least partly due to a better retention of protein-bound lipids.

Bacterial Outer Membrane Proteins↗

PubMed2003

Membrane protein folding: beyond the two stage model.

The folding of alpha-helical membrane proteins has previously been described using the two stage model, in which the membrane insertion of independently stable alpha-helices is followed by their mutual interactions within the membrane to give higher order folding and oligomerization. Given recent advances in our understanding of membrane protein structure it has become apparent that in some cases the model may not fully represent the folding process. Here we present a three stage model which gives considerations to ligand binding, folding of extramembranous loops, insertion of peripheral domains and the formation of quaternary structure.

Aquaporins↗

PubMed2003

An atypical haem in the cytochrome b(6)f complex.

Photosystems I and II (PSI and II) are reaction centres that capture light energy in order to drive oxygenic photosynthesis; however, they can only do so by interacting with the multisubunit cytochrome b(6)f complex. This complex receives electrons from PSII and passes them to PSI, pumping protons across the membrane and powering the Q-cycle. Unlike the mitochondrial and bacterial homologue cytochrome bc(1), cytochrome b(6)f can switch to a cyclic mode of electron transfer around PSI using an unknown pathway. Here we present the X-ray structure at 3.1 A of cytochrome b(6)f from the alga Chlamydomonas reinhardtii. The structure bears similarities to cytochrome bc(1) but also exhibits some unique features, such as binding chlorophyll, beta-carotene and an unexpected haem sharing a quinone site. This haem is atypical as it is covalently bound by one thioether linkage and has no axial amino acid ligand. This haem may be the missing link in oxygenic photosynthesis.

Animals↗

PubMed2003

Slow diffusion of macromolecular assemblies by a new pulsed field gradient NMR method.

The translational diffusion coefficient of an integral membrane protein/surfactant complex has been measured using a novel pulsed field gradient NMR method. In this new approach, the information about the localization of the molecules is temporarily stored in the form of longitudinal magnetization of isotopes with long spin-lattice relaxation times. This allows one to increase the duration of the diffusion interval by about 1 order of magnitude. Unlike standard proton NMR methods using pulsed field gradients and stimulated echoes, the new method can be applied to macromolecular assemblies with diffusion coefficients well below 10(-10) m(2) s(-1), corresponding to masses in excess of 25 kDa in aqueous solution at room temperature. The method was illustrated by application to a water-soluble complex of tOmpA, the hydrophobic transmembrane domain of bacterial outer membrane protein A, with the detergent octyl-tetraoxyethylene (C(8)E(4); overall mass of complex approximately 45 kDa). The diffusion coefficient was found to be D = (4.99 +/- 0.07) x 10(-11) m(2) s(-1), consistent with measurements by size exclusion chromatography and by ultracentrifugation. The method has also been applied to a solution of recombinant human tRNA(3)(Lys), which has a molecular mass of 24 kDa, and the diffusion coefficient D = (1.05 +/- 0.015) x 10(-10) m(2) s(-1).

Bacterial Outer Membrane Proteins↗

PubMed2003

Site-directed photochemical coupling of cytochrome b6f-associated chlorophyll.

Cytochrome b(6)f complexes contain a molecule of chlorophyll a (Chla), which, in Chlamydomonas reinhardtii, can be exchanged for extraneous chlorophyll during protracted incubation of the purified complex in detergent solution. The specificity of the site and its location in the complex have been studied by photochemical coupling and circular dichroism spectroscopy. Following substitution of the original chlorophyll with [(3)H]Chla, the complex was irradiated in the Soret absorption band of Chla to complete bleaching and the amount of radioactivity covalently bound to each b(6)f subunit determined. Strong labeling was found to be associated with cytochrome f. The labeling originates from [(3)H]Chla molecules bound to a slowly exchanging site and showing the properties of the endogenous Chl, not from molecules dissolved in the detergent belt surrounding the complex. Chlorophyll b (Chlb) can compete with Chla, albeit with a lower affinity. Irradiation of [(3)H]Chlb introduced into the slowly exchanging site yielded the same labeling pattern that was observed with [(3)H]Chla. Proteolytic cleavage showed [(3)H]Chla labeling to be strictly restricted to the C-terminal region of cytochrome f. Circular dichroism spectra of the native complex revealed a bilobed signal characteristic of excitonic interaction between chlorophylls. The structural and evolutionary implications of these findings are discussed.

Animals↗

PubMed2002

Allosteric transitions of Torpedo acetylcholine receptor in lipids, detergent and amphipols: molecular interactions vs. physical constraints.

The binding of a fluorescent agonist to the acetycholine receptor from Torpedo electric organ has been studied by time-resolved spectroscopy in three different environments: in native membrane fragments, in the detergent CHAPS, and after complexation by amphipathic polymers ('amphipols'). Binding kinetics was similar in the membrane and in amphipols, demonstrating that the receptor can display unaltered allosteric transitions outside its natural lipid environment. In contrast, allosteric equilibria were strongly shifted towards the desensitized state in CHAPS. Therefore, the effect of CHAPS likely results from molecular interactions rather than from the loss of bulk physical properties of the membrane environment.

Allosteric Regulation↗

PubMed2002

Synthesis and preliminary biochemical assessment of ethyl-terminated perfluoroalkylamine oxide surfactants.

The synthesis and usefulness in membrane biochemistry of a new class of surfactants have been investigated. 1-Ethyl-2-dimethylamine oxide polar heads were grafted onto a hydrocarbon, a fluorocarbon or an ethyl-capped fluorocarbon hydrophobic tail. The ability of the resulting surfactants to extract and/or to stabilize in aqueous solution a test membrane protein, cytochrome b(6)f, was evaluated. While it is not a detergent, the hemifluorinated derivative efficiently kept purified cytochrome b(6)f soluble, native and functional. The data suggest that alkyl-capped fluorocarbon surfactants provide an interesting alternative to classical detergents for handling membrane proteins in aqueous solutions under non-dissociating conditions.

Cytochrome b Group↗

PubMed2002

Chimeric fusions of subunit IV and PetL in the b6f complex of Chlamydomonas reinhardtii: structural implications and consequences on state transitions.

The cytochrome b(6)f complex of Chlamydomonas reinhardtii contains four large subunits and at least three small ones, PetG, PetL, and PetM, whose role and location are unknown. Chimeric proteins have been constructed, in which the C terminus of subunit IV is fused to either one or the other of the two putative N termini of PetL. Biochemical and functional analysis of the chimeras together with mass spectrometry analysis of the wild-type (WT) complex led to the following conclusions: (i) neither a free subunit IV C terminus nor a free PetL N terminus is required for assembly of the b(6)f complex; (ii) the first AUG codon in the sequence of the gene petL is used for initiation; (iii) the N terminus of WT PetL lies in the lumen; (iv) in the WT complex, the N terminus of PetL and the C terminus of subunit IV are within reach of each other; (v) the purified b(6)f complex from C. reinhardtii contains an eighth, hitherto unrecognized subunit, PetN; and (vi) the ability to perform state transitions is lost in the chimeric mutants, although (vii) the Q-cycle is unaffected. A structural hypothesis is presented to account for this peculiar phenotype.

Amino Acid Sequence↗