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BBA - Biomembranes (v.1716, #1)
Computational analysis of mutations in the transmembrane region of Vpu from HIV-1 by Andrew Candler; Matthew Featherstone; Rehan Ali; Leslie Maloney; Anthony Watts; Wolfgang B. Fischer (pp. 1-10).
Vpu is an 81 amino acid integral membrane protein encoded by HIV-1. Its α-helical transmembrane (TM) domain (residues ∼6–28) enhances virion release by oligomerizing into bundles and forming ion-conducting channels across the plasma membrane. Its cytoplasmic domain (residues ∼29–81) is also α-helical and binds to the transmembrane protein CD4, inducing its degradation. Mutations within the TM domain have been found to abrogate enhanced particle release from the infected cell (Tiganos et al. Virology (1998) 251 96–107). A series of computational models of monomeric, pentameric and hexameric Vpu1–31 mutants have been constructed, embedded in fully hydrated lipid bilayers and subjected to a 3 ns molecular dynamics (MD) simulation. None of the mutations has any destabilizing effect on the secondary and tertiary structure. One of the mutants, in which the position of a tryptophan residue within the TM domain is altered, is known not to induce CD4 degradation; an extended kinked model of this mutant has been generated (Vpu1–52IVW-k) and during subsequent MD simulations, the bend between the TM and a part of the cytoplasmic domain is found to unwind and a complex salt bridge involving Lys-37 is formed.
Keywords: Vpu; HIV-1; Mutant; Viral membrane protein; Molecular dynamics simulation; protein structure
Membrane-disruptive abilities of β-hairpin antimicrobial peptides correlate with conformation and activity: A31P and1H NMR study by Rajeswari Mani; Alan J. Waring; Robert I. Lehrer; Mei Hong (pp. 11-18).
The membrane interaction and solution conformation of two mutants of the β-hairpin antimicrobial peptide, protegrin-1 (PG-1), are investigated to understand the structural determinants of antimicrobial potency. One mutant, [A6,8,13,15] PG-1, does not have the two disulfide bonds in wild-type PG-1, while the other, [Δ4,18 G10] PG-1, has only half the number of cationic residues.31P solid-state NMR lineshapes of uniaxially aligned membranes indicate that the membrane disorder induced by the three peptides decreases in the order of PG-1>[Δ4,18 G10] PG-1≫[A6,8,13,15] PG-1. Solution NMR studies of the two mutant peptides indicate that [Δ4,18 G10] PG-1 preserves the β-hairpin fold of the wild-type peptide while [A6,8,13,15] PG-1 adopts a random coil conformation. These NMR results correlate well with the known activities of these peptides. Thus, for this class of peptides, the presence of a β-hairpin fold is more essential than the number of cationic charges for antimicrobial activity. This study indicates that31P NMR lineshapes of uniaxially aligned membranes are well correlated with antimicrobial activity, and can be used as a diagnostic tool to understand the peptide–lipid interactions of these antimicrobial peptides.
Keywords: Abbreviations; PG-1; protegrin-1; POPC; 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine; POPG; 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylglycerol; POPE; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine; DLPC; dilauroylphosphatidylcholine; DMPC; dimyristoylphosphatidylcholine; DPG; diphosphatidylglycerolAntimicrobial peptide; PG-1; 31; P solid-state NMR; Conformation; Membrane disorder; Solution NMR
Effect of ischemia reperfusion on sodium-dependent phosphate transport in renal brush border membranes by Syed J. Khundmiri; Mohammed Asghar; Anees A. Banday; Farah Khan; Samina Salim; Moshe Levi; Ahad N.K. Yusufi (pp. 19-28).
The effect of ischemia induced acute renal failure (ARF) on the transport of phosphate (Pi) after early (15–30 min) and prolonged (60 min) ischemia in the brush border membrane vesicles (BBMV) from rat renal cortex was studied. Sodium-dependent transport of Pi declined significantly and progressively due to ischemia. Western blot analysis of BBM from ischemic rats showed decreased expression of NaPi-2. A compensatory increase was observed in Pi uptake in BBMV from contralateral kidneys. There was no significant difference in NaPi-2 expression between BBMV from sham and contralateral kidneys. Early blood reperfusion for 15 min after 30 min ischemia caused further decline in Pi uptake. Prolonged reperfusion for 120 min caused partial reversal of transport activities in 30-min ischemic rats. However, no improvement in the transport of Pi was observed in 60-min ischemic rats after 120 min of blood reperfusion. Kinetic studies showed that the effect of ischemia and blood reperfusion was dependent on the Vmax of the Na-Pi transporter. Western blot analysis showed increased expression of NaPi-2 in the BBMs from ischemia–reperfusion animals. Further, a shift in the association of Na ions to transport one molecule of Pi was observed under different extracellular Na concentrations [Na]o. Feeding rats with low Pi diet and/or treatment with thyroid hormone (T3) prior to ischemia resulted in increased basal Pi transport. Ischemia caused similar decline in Pi transport in BBM from LPD and/or T3 animals. However, recovery in these animals was faster than the normal Pi diet fed (NPD) animals. The study suggests a change in the intrinsic properties of the Na-Pi transporter in rat kidneys due to ischemia. The study also indicates that treatment with T3 and feeding LPD prior to ischemia caused faster recovery of phosphate uptake due to ischemia–reperfusion injury.
Keywords: Acute renal failure; Ischemia; Pi transport; L-Proline transport; Thyroid hormone; Low phosphorus diet
Investigation of a novel artificial antimicrobial peptide by fluorescence correlation spectroscopy: An amphipathic cationic pattern is sufficient for selective binding to bacterial type membranes and antimicrobial activity by Lanlan Yu; Jeak Ling Ding; Bow Ho; Thorsten Wohland (pp. 29-39).
Fluorescence Correlation Spectroscopy (FCS) is used to study the interaction of a recently designed antimicrobial peptide, called V4, with LPS and lipids of varying head and tail groups. V4 is designed based on a known amphipathic cationic pattern BHPHB (B: basic; H: hydrophobic; P: polar residue, respectively) and shows a good combination of high antimicrobial activity, low cytotoxic activity and low hemolytic activity. It is shown that V4 has high binding affinity for LPS, which is the major component of the outer membrane of Gram-negative bacteria, and shows selectivity for negatively charged lipids in contrast to zwitterionic lipids at a low peptide/lipid ratio. At high peptide/lipid ratio, V4 can permeabilize vesicles composed of negatively charged lipids and eventually cause vesicle fusion. The identification of the amphipathic cationic pattern as the mediator of selectivity and antimicrobial activity could be a first step in the rational design of better antimicrobial peptides.
Keywords: Abbreviations; V4; CVKVQVKVGSGVKVQVKVC; Rho 6G; Rhodamine 6G chloride; TMR; tetramethylrhodamine; R18; octadecyl rhodamine B chloride; LPS; lipo-polysaccharide; FITC-LPS; fluorescein isothiocyanate lipopolysaccharide; PBS; Phosphate buffered saline; PC; Phosphatidylcholine; DPPC; 1,2-Dipalmitoyl-; sn; -Glycero-3-Phosphocholine; DPPE; 1,2-Dipalmitoyl-; sn; -Glycero-3-Phosphoethanolamine; DPPG; 1,2-Dipalmitoyl-; sn; -Glycero-3-[Phospho-rac-(1-glycerol)]; POPC; 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine; POPE; 1-Palmitoyl-2-Oleoyl-; sn; -Glycero-3-Phospho-ethanolamine; POPG; 1-Palmitoyl-2-Oleoyl-; sn; -Glycero-3-[Phospho-rac-(1-glycerol)]; Rho-PE; 1,2-Dipalmitoyl-; sn; -Glycero-3-Phospho-ethanolamine-N-(Lissamine Rhodamine B Sulfonyl) (Ammonium Salt)Antimicrobial peptide; Fluorescence correlation spectroscopy
Composition dependence of vesicle morphology and mixing properties in a bacterial model membrane system by B. Pozo Navas; K. Lohner; G. Deutsch; E. Sevcsik; K.A. Riske; R. Dimova; P. Garidel; G. Pabst (pp. 40-48).
We have determined the mixing properties and lamellar organization of bacterial membrane mimetics composed of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) and -phosphatidylglycerol (POPG) at various molar ratios applying differential scanning calorimetry, small and wide-angle X-ray scattering, as well as optical phase contrast microscopy. Combining the experimental thermodynamic data with a simulation of the liquidus and solidus lines, we were able to construct a phase diagram. Using this approach, we find that the lipids mix in all phases non-ideally in the thermodynamic sense. As expected, pure POPE assembles into multilamellar and pure POPG into unilamellar vesicles, respectively, which are stable within the studied temperature range. In contrast, mixtures of the two components form oligolamellar vesicles consisting of about three to five bilayers. The layers within these oligolamellar liposomes are positionally correlated within the gel phase, but become uncorrelated within the fluid phase exhibiting freely fluctuating bilayers, while the vesicles as a whole remain intact and do not break up into unilamellar forms. X-ray, as well as DSC data, respectively, reveal a miscibility gap due to a lateral phase segregation at POPG concentrations above about 70 mol%, similar to previously reported data on mixtures composed of disaturated PEs and PGs. Hence, the existence of a region of immiscibility is a general feature of PE/PG mixtures and the mixing properties are dominated by PE/PG headgroup interactions, but are largely independent of the composition of the hydrocarbon chains. This is in accordance with a recent theoretical prediction.
Keywords: Phase diagram; Phase separation; Liposome; X-ray diffraction; Differential scanning calorimetry; Optical microscopy
Coverage-dependent changes of cytochrome c transverse location in phospholipid membranes revealed by FRET by Yegor A. Domanov; Julian G. Molotkovsky; Galyna P. Gorbenko (pp. 49-58).
The method of fluorescence resonance energy transfer (FRET) has been employed to monitor cytochrome c interaction with bilayer phospholipid membranes. Liposomes composed of phosphatidylcholine and varying amounts of anionic lipid cardiolipin (CL) were used as model membranes. Trace amount of fluorescent lipid derivative, anthrylvinyl-phosphatidylcholine was incorporated into the membranes to serve energy donor for heme moiety of cytochrome c. Energy transfer efficiency was measured at different lipid and protein concentrations to obtain extensive set of data, which were further analyzed globally in terms of adequate models of protein adsorption and energy transfer on the membrane surface. It has been found that the cytochrome c association with membranes containing 10 mol% CL can be described in terms of equilibrium binding model (yielding dissociation constant Kd=0.2–0.4 μM and stoichiometry n=11–13 lipid molecules per protein binding site) combined with FRET model assuming uniform acceptor distribution with the distance of 3.5–3.6 nm between the bilayer midplane and heme moiety of cytochrome c. However, increasing the CL content to 20 or 40 mol% (at low ionic strength) resulted in a different behavior of FRET profiles, inconsistent with the concepts of equilibrium adsorption of cytochrome c at the membrane surface and/or uniform acceptor distribution. To explain this fact, several possibilities are analyzed, including cytochrome c-induced formation of non-bilayer structures and clusters of charged lipids, or changes in the depth of cytochrome c penetration into the bilayer depending on the protein surface density. Additional control experiments have shown that only the latter process can explain the peculiar concentration dependences of FRET at high CL content.
Keywords: Cytochrome; c; FRET; Membrane insertion; Quantitative analysis; Cardiolipin
Using micellar mole fractions to assess membrane protein stability in mixed micelles by P. Sehgal; J.E. Mogensen; D.E. Otzen (pp. 59-68).
The increased focus on the structural and physical properties of membrane proteins has made it critical to develop methods that provide a reliable estimate of membrane protein stability. A simple approach is to monitor the protein's conformational changes in mixed detergent systems, typically consisting of an anionic (denaturing) and non-ionic (non-denaturing) component. Linear correlations between, e.g., the melting temperature and the bulk mole fraction of the anionic component have been observed. However, a potential complication is that the bulk mole fraction is not identical to the mole fraction in the mixed micelle, which is the local environment experienced by the membrane protein. Here, we present an extensive analysis of the thermal stability of the membrane-integrated domain of the outer membrane protein AIDA in the presence of different mixed micelles. In the micelle system SDS-octyl-polyoxyethylene, the melting temperature in the absence of SDS extrapolates to 113 °C using bulk mole fractions. However, for mixed micelles involving short-chain detergents or phospholipids, the melting temperature calculated using bulk mole fractions reaches values up to several hundred degrees higher than 113 °C and can only be obtained by extrapolation over a narrow mole fraction interval. Furthermore, there is a non-linear relationship between the melting temperature and bulk mole fractions for mixed micelle systems involving cationic detergents (also denaturing). We show that if we instead use the micellar mole fraction as a parameter for denaturing detergent strength, we obtain linear correlations which extrapolate to more or less the same value of the melting temperature. There remains some scatter in the extrapolated values of the melting temperature in different binary systems, which suggest that additional micellar interactions may play a role. Nevertheless, in general terms, the mixed micellar composition is a good parameter to describe the membrane protein's microenvironment. Note, however, that for the mixed micelle system involving SDS and dodecyl maltoside, which has been used by several research groups to determine membrane protein stability, the estimate provided by bulk mole fraction leads to similar values as that of micellar mole fractions.
Keywords: Abbreviations; α; i; bulk mole fraction of ionic detergent; α; n; bulk mole fraction of non-ionic detergent; AIDA; residues 951–1286 of Adhesin Involved in Diffuse Adherence; DCPC; 1,2-dicapryl-; sn; -glycero-3-phosphocholine; DecM; n-decyl-β-; d; -maltoside; DHPC; 1,2-diheptanoyl-; sn; -glycero-3-phosphocholine; DLPC; 1,2-dilauroyl-; sn; -glycero-3-phosphocholine; DM; n-dodecyl-β-; d; -maltoside; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; LTAC; lauroyl trimethyl ammonium chloride; NM; n-nonyl-β-; d; -maltoside; NPN; N-phenyl-1-napthylamine; OG; n-octyl-β-; d; -glucoside; OM; n-octyl-β-; d; -maltoside; oPOE; octyl-polyoxyethylene; SDeS; sodium decyl sulfate; SDS; sodium dodecyl sulfate; SHS; sodium hexadecyl sulfate; STS; sodium tetradecyl sulfate; T; m; melting temperature; UM; n-undecyl-β-; d; -maltoside; X; i; micellar mole fraction of ionic detergent; X; n; micellar mole fraction of non-ionic detergentOuter membrane protein; Detergents; Mixed micelle; Thermal stability; Unfolding kinetics; Micellar composition
Large-scale purification of the proton pumping pyrophosphatase from Thermotoga maritima: A “Hot-Solveâ€? method for isolation of recombinant thermophilic membrane proteins by Rosa L. López-Marqués; José R. Pérez-Castiñeira; Morten J. Buch-Pedersen; Sergio Marco; Jean-Louis Rigaud; Michael G. Palmgren; Aurelio Serrano (pp. 69-76).
Although several proton-pumping pyrophosphatases (H+-PPases) have been overexpressed in heterologous systems, purification of these recombinant integral membrane proteins in large amounts in order to study their structure–function relationships has proven to be a very difficult task. In this study we report a new method for large-scale production of pure and stable thermophilic H+-PPase from Thermotoga maritima. Following overexpression in yeast, a “Hot-Solve� procedure based on high-temperature solubilization and metal-affinity chromatography was used to obtain a highly purified detergent-solubilized TVP fraction with a yield around 1.5 mg of protein per litre of yeast culture. Electron microscopy showed the monodispersity of the purified protein and single particle analysis provided the first direct evidence of a dimeric structure for H+-PPases. We propose that the method developed could be useful for large-scale purification of other recombinant thermophilic membrane proteins.
Keywords: Abbreviations; PPi; inorganic pyrophosphate; H; +; -PPase; proton-pumping pyrophosphatase; DTT; ditiothreitol; DDM; n-dodecyl-β-; d; -maltopyranoside; TVP; Thermotoga maritima; membrane-bound H; +; -PPase; PMSF; phenyl-methyl-sulphonyl-fluoride; MES; 2-morpholinoethansulfonic acid; NTA; nitriloacetic acid; Tris; Tris-(hydroxylmethyl) aminomethane; EM; electron microscopy; SOM; self-organizing mappingH; +; -PPase; Thermotoga maritima; Ni-NTA affinity chromatography; Detergent solubilization; Single particle analysis