Cardiolipin vesicles can accommodate cholesterol up to 0.80 mole fraction, i.e. one molecule per cardiolipin fatty acid chain

AbstractCardiolipin-cholesterol interactions were studied by time-resolved fluorescence spectroscopy with 1,6-diphenyl-1, 3,5-hexatriene as probe. The residual anisotropy parameter, r∞ (reflecting the fatty acid chain packing), was measured in the liquid crystalline phase as a function of cholesterol addition. Two main results are reported: (i) a slight increase of the order parameter, S, computed from the r∞ value as S = (r∞/r0)12, in the physiological concentration range of cholesterol; (ii) a sharp enhancement of S from a cholesterol mole fraction (X̄chl) of 0.20 and up to X̄chl of 0.80. This is in contrast to unsaturated kcithin systems for which a continuous increase of the order parameter was monitored, culminating at X̄chl = 0.50, the wellknown maximum level of incorporation of cholesterol into lecithin model membranes

Adenylate cyclase of bovine adrenal cortex plasma membranes Divergence between corticotropin and fluoride combined effects with forskolin

AbstractThe diterpene forskolin maximally stimulated bovine adrenal cortex adenylate cyclase activity 9-fold with a concentration producing half-maximum effect (ED50) of about 4 μM. The effects of forskolin and the fully active corticotropin fragment ACTH (1–24) were additive over nearly the whole range of concentration of both effectors, indicating separate and independent mechanisms of action. By contrast, 10 mM NaF blocked forskolin action in the nanomolar range of the diterpene concentration, while it allowed a partial stimulation by forskolin in the micromolar range. NaF thus reveals a heterogeneity of forskolin action in the adrenal cortex plasma membranes. Moreover, our data suggest that ACTH and NaF activation effects, both mediated by the stimulatory regulatory protein Gs, proceed through different mechanisms

Bindings of hMRP1 transmembrane peptides with dodecylphosphocholine and dodecyl-β-d-maltoside micelles: A molecular dynamics simulation study

International audienceIn this paper, we describe molecular dynamics simulation results of the interactions between four peptides (mTM10, mTM16, TM17 and KTM17) with micelles of dodecylphosphocholine (DPC) and dodecyl-β-Dmaltoside (DDM). These peptides represent three transmembrane fragments (TM10, 16 and 17) from the MSD1 and MSD2 membrane-spanning domains of an ABC membrane protein (hMRP1), which play roles in the protein functions. The peptide–micelle complex structures, including the tryptophan accessibility and dynamics were compared to circular dichroism and fluorescence studies obtained in water, trifluoroethanol and with micelles. Our work provides additional results not directly accessible by experiments that give further support to the fact that these peptides adopt an interfacial conformation within the micelles. We also show that the peptides are more buried in DDM than in DPC, and consequently, that they have a larger surface exposure to water in DPC than in DDM. As noted previously by simulations and experiments we have also observed formation of cation–π bonds between the phosphocholine DPC headgroup and Trp peptide residue. Concerning the peptide secondary structures (SS), we find that in TFE their initial helical conformations are maintained during the simulation, whereas in water their initial SS are lost after few nanoseconds of simulation. An intermediatesituation is observed with micelles, where the peptides remain partially folded and more structured in DDMthan in DPC. Finally, our results show no sign of β-strand structure formation as invoked by far-UV CDexperiments even when three identical peptides are simulated either in water or with micelle

Single-spanning membrane protein insertion in membrane mimetic systems: role and localization of aromatic residues.

International audienceMembrane protein insertion in the lipid bilayer is determining for their activity and is governed by various factors such as specific sequence motifs or key amino-acids. A detailed fluorescence study of such factors is exemplified with PMP1, a small (38 residues) single-membrane span protein that regulates the plasma membrane H(+)-ATPase in yeast and specifically interacts with phosphatidylserines. Such interactions may stabilize raft domains that have been shown to contain H(+)-ATPase. Previous NMR studies of various fragments have focused on the critical role of interfacial residues in the PMP1 structure and intermolecular interactions. The C-terminal domain contains a terminal Phe (F38), a single Trp (W28) and a single Tyr (Y25) that may act together to anchor the protein in the membrane. In order to describe the location and dynamics of W28 and the influence of Y25 on protein insertion within membrane, we carried out a detailed steady-state and time-resolved fluorescence study of the synthetic G13-F38 fragment and its Tyr-less mutant, Y25L in various membrane mimetic systems. Detergent micelles are conveniently used for this purpose. We used dodecylphosphocholine (DPC) in order to compare with and complement previous NMR results. In addition, dodecylmaltoside (DM) was used so that we could apply our recently described new quenching method by two brominated analogs of DM (de Foresta et al. 2002, Eur. Biophys. J. 31:185-97). In both systems, and in the presence and absence of Y25, W28 was shown to be located below but close to the polar headgroup region, as shown by its maximum emission wavelengths (lambda(max)), curves for the quenching of Trp by the brominated analogs of DM and bimolecular constants for quenching (k(q)) by acrylamide. Results were interpreted by comparison with calibration data obtained with fluorescent model peptides. Time-resolved anisotropy measurements were consistent with PMP1 fragment immobilization within peptide-detergent complexes. We tentatively assigned the two major Trp lifetimes to the Trp (chi(1)=60 degrees and 180 degrees ) rotamers, based on the recent lifetime-rotamer correlation proposed for model cyclic peptides (Pan and Barkley 2004, Biophys J 86:3828-35). We also analyzed the role of the hydrophobic anchor, by comparing the micelle binding of fragments of various lengths including the synthesized full-length protein and detected peculiar differences for protein interaction with the polar headgroups of DM or DPC