Proton Motive Force-Dependent Hoechst 33342 Transport by the ABC Transporter LmrA of Lactococcus lactis

The fluorescent compound Hoechst 33342 is a substrate for many multidrug resistance (MDR) transporters and is widely used to characterize their transport activity. We have constructed mutants of the adenosine triphosphate (ATP) binding cassette (ABC)-type MDR transporter LmrA of Lactococcus lactis that are defective in ATP hydrolysis. These mutants and wild-type LmrA exhibited an atypical behavior in the Hoechst 33342 transport assay. In membrane vesicles, Hoechst 33342 transport was shown to be independent of the ATPase activity of LmrA, and it was not inhibited by orthovanadate but sensitive to uncouplers that collapse the proton gradient and to N,N'-dicyclohexylcarbodiimide, an inhibitor of the F0F1-ATPase. In contrast, transport of Hoechst 33342 by the homologous, heterodimeric MDR transporter LmrCD showed a normal ATP dependence and was insensitive to uncouplers of the proton gradient. With intact cells, expression of LmrA resulted in an increased rate of Hoechst 33342 influx while LmrCD caused a decrease in the rate of Hoechst 33342 influx. Cellular toxicity assays using a triple knockout strain, i.e., L. lactis ΔlmrA ΔlmrCD, demonstrate that expression of LmrCD protects cells against the growth inhibitory effects of Hoechst 33342, while in the presence of LmrA, cells are more susceptible to Hoechst 33342. Our data demonstrate that the LmrA-mediated Hoechst 33342 transport in membrane vesicles is influenced by the transmembrane pH gradient due to a pH-dependent partitioning of Hoechst 33342 into the membrane.

Le middleware OSA+ (une architecture micronoyau pour temps réel)

STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Self-Association Process of a Peptide in Solution: From β-Sheet Filaments to Large Embedded Nanotubes

Lanreotide is a synthetic octapeptide used in the therapy against acromegaly. When mixed with pure water at 10% (w/w), Lanreotide (acetate salt) forms liquid crystalline and monodisperse nanotubes with a radius of 120 Å. The molecular and supramolecular organization of these structures has been determined in a previous work as relying on the lateral association of 26 β-sheet filaments made of peptide noncovalent dimers, the basic building blocks. The work presented here has been devoted to the corresponding self-association mechanisms, through the characterization of the Lanreotide structures formed in water, as a function of peptide (acetate salt) concentration (from 2% to 70% (w/w)) and temperature (from 15°C to 70°C). The corresponding states of water were also identified and quantified from the thermal behavior of water in the Lanreotide mixtures. At room temperature and below 3% (w/w) Lanreotide acetate in water, soluble aggregates were detected. From 3% to 20% (w/w) long individual and monodisperse nanotubes crystallized in a hexagonal lattice were evidenced. Their molecular and supramolecular organizations are identical to the ones characterized for the 10% (w/w) sample. Heating induces the dissolution of the nanotubes into soluble aggregates of the same structural characteristics as the room temperature ones. The solubilization temperature increases from 20°C to 70°C with the peptide concentration and reaches a plateau between 15% and 25% (w/w) in peptide. These aggregates are proposed to be the β-sheet filaments that self-associate to build the walls of the nanotubes. Above 20% (w/w) of Lanreotide acetate in water, polydisperse embedded nanotubes are formed and the hexagonal lattice is lost. These embedded nanotubes exhibit the same molecular and supramolecular organizations as the individual monodisperse nanotubes formed at lower peptide concentration. The embedded nanotubes do not melt in the range of temperature studied indicating a higher thermodynamic stability than individual nanotubes. In parallel, the thermal behaviors of water in mixtures containing 2–80% (w/w) in peptide have been studied by differential scanning calorimetry, and three different types of water were characterized: 1), bulk water melting at 0°C, 2), nonfreezing water, and 3), interfacial water melting below 0°C. The domains of existence and coexistence of these different water states are related to the different Lanreotide supramolecular structures. All these results were compiled into a binary Lanreotide-water phase diagram and allowed to propose a self-association mechanism of Lanreotide filaments into monodisperse individual nanotubes and embedded nanotubes

Functional reconstitution of the nicotinic acetylcholine receptor by CHAPS dialysis depends on the concentrations of salt, lipid, and protein

Schürholz T, Kehne J, Gieselmann A, Neumann E. Functional reconstitution of the nicotinic acetylcholine receptor by CHAPS dialysis depends on the concentrations of salt, lipid, and protein. Biochemistry. 1992;31(21):5067-5077.The detergent CHAPS was found to be the preferable surfactant for the efficient purification and reconstitution of the Torpedo californica nicotinic acetylcholine receptor (AChR). The main result is that the incorporation of the AChR proteins into lipid vesicles by CHAPS dialysis was strongly dependent on the salt and protein concentrations. As monitored by sucrose gradients, by electron microscopy, and by agonist-induced lithium ion flux, the best reconstitution yields were obtained in 0.5 M NaCl at a protein concentration of 0.5 g/L and in 0.84 M NaCl at 0.15 g/L protein. Electron micrographs of receptor molecules, which were incorporated into vesicles, showed single, nonaggregated dimer (M(r) = 580 000) and monomer (M(r) = 290 000) species. CHAPS dialysis at NaCl concentrations < 0.5 M largely reduced the receptor incorporation concomitant with protein aggregation. Electron micrographs of these preparations revealed large protein sheets or ribbons not incorporated into vesicles. The analysis of static and dynamic light scattering demonstrated that the detergent-solubilized AChR molecules aggregate at low lipid contents (less-than-or-equal-to 500 phospholipids/AChR dimer), independent of the salt concentration. AChR proteins eluted from an affinity column with a solution containing 8 mM CHAPS (but no added lipid) still contained 130 +/- 34 tightly bound phospholipids per dimer. The aggregates (about 10 dimers on the average) could be dissociated by readdition of lipid and, interestingly, also by increasing the CHAPS concentration up to 15 mM. This value is much higher than the CMC of CHAPS = 4.0 +/- 0.4 mM, which was determined by surface tension measurements. The data clearly suggest protein-micelle interactions in addition to the association of monomeric detergents with proteins. Furthermore, the concentration of the (free) monomeric CHAPS at the vesicle-micelle transformation in 0.5 M NaCl ([D(W)]c = 3.65 mM) was higher than in 50 mM NaCl ([D(W)]c = 2.8 mM). However, it is suggested that the main effect of high salt concentrations during the reconstitution process is an increase of the fusion (rate) of the ternary protein/lipid/CHAPS complexes with mixed micelles or with vesicular structures, similar to the salt-dependent fusion of vesicles

Leakage and lysis of lipid membranes induced by the lipopeptide surfactin

Surfactin is a lipopeptide produced by Bacillus subtilis which possesses antimicrobial activity. We have studied the leakage and lysis of POPC vesicles induced by surfactin using calcein fluorescence de-quenching, isothermal titration calorimetry and (31)P solid state NMR. Membrane leakage starts at a surfactin-to-lipid ratio in the membrane, R (b) approximately 0.05, and an aqueous surfactin concentration of C (S) (w) approximately 2 microM. The transient, graded nature of leakage and the apparent coupling with surfactin translocation to the inner leaflet of the vesicles, suggests that this low-concentration effect is due to a bilayer-couple mechanism. Different permeabilization behaviour is found at R (b) approximately 0.15 and attributed to surfactin-rich clusters, which can induce leaks and stabilize them by covering their hydrophobic edges. Membrane lysis or solubilization to micellar structures starts at R (b) (sat) = 0.22 and C (S) (w) = 9 microM and is completed at R (m) (sol) = 0.43 and C (S) (w) = 11 microM. The membrane-water partition coefficient of surfactin is obtained as K = 2 x 10(4) M(-1). These data resolve inconsistencies in the literature and shed light on the variety of effects often referred to as detergent-like effects of antibiotic peptides on membranes. The results are compared with published parameters characterizing the hemolytic and antibacterial activity

Atomic view of the histidine environment stabilizing higher-pH conformations of pH-dependent proteins

External stimuli are powerful tools that naturally control protein assemblies and functions. For example, during viral entry and exit changes in pH are known to trigger large protein conformational changes. However, the molecular features stabilizing the higher pH structures remain unclear. Here we elucidate the conformational change of a self-assembling peptide that forms either small or large nanotubes dependent on the pH. The sub-angstrom high-pH peptide structure reveals a globular conformation stabilized through a strong histidine-serine H-bond and a tight histidine-aromatic packing. Lowering the pH induces histidine protonation, disrupts these interactions and triggers a large change to an extended β-sheet-based conformation. Re-visiting available structures of proteins with pH-dependent conformations reveals both histidine-containing aromatic pockets and histidine-serine proximity as key motifs in higher pH structures. The mechanism discovered in this study may thus be generally used by pH-dependent proteins and opens new prospects in the field of nanomaterials