Incorporation of outer membrane protein OmpG in lipid membranes: protein-lipid interactions and beta-barrel orientation

OmpG is an intermediate size, monomeric, outer membrane protein from Escherichia coli, with n beta = 14 beta-strands. It has a large pore that is amenable to modification by protein engineering. The stoichiometry ( N b = 20) and selectivity ( K r = 0.7-1.2) of lipid-protein interaction with OmpG incorporated in dimyristoyl phosphatidylcholine bilayer membranes was determined with various 14-position spin-labeled lipids by using EPR spectroscopy. The limited selectivity for different lipid species is consistent with the disposition of charged residues in the protein. The conformation and orientation (beta-strand tilt and beta-barrel order parameters) of OmpG in disaturated phosphatidylcholines of odd and even chain lengths from C(12:0) to C(17:0) was determined from polarized infrared spectroscopy of the amide I and amide II bands. A discontinuity in the protein orientation (deduced from the beta-barrel order parameters) is observed at the point of hydrophobic matching of the protein with lipid chain length. Compared with smaller (OmpA; n beta = 8) and larger (FhuA; n beta = 22) monomeric E. coli outer membrane proteins, the stoichiometry of motionally restricted lipids increases linearly with the number of beta-strands, the tilt (beta approximately 44 degrees ) of the beta-strands is comparable for the three proteins, and the order parameter of the beta-barrel increases regularly with n beta. These systematic features of the integration of monomeric beta-barrel proteins in lipid membranes could be useful for characterizing outer membrane proteins of unknown structure.     

Protein-lipid interactions with Fusobacterium nucleatum major outer membrane protein FomA: spin-label EPR and polarized infrared spectroscopy

FomA, the major outer membrane protein of Fusobacterium nucleatum, was expressed and purified in Escherichia coli and reconstituted from detergent in bilayer membranes of phosphatidylcholines with chain lengths from C(12:0) to C(17:0). The conformation and orientation of membrane-incorporated FomA were determined from polarized, attenuated total reflection, infrared (IR) spectroscopy, and lipid-protein interactions with FomA were characterized by using electron paramagnetic resonance (EPR) spectroscopy of spin-labeled lipids. Approximately 190 residues of membranous FomA are estimated to be in a beta-sheet configuration from IR band fitting, which is consistent with a 14-strand transmembrane beta-barrel structure. IR dichroism of FomA indicates that the beta-strands are tilted by approximately 45 degrees relative to the sheet/barrel axis and that the order parameter of the latter displays a discontinuity corresponding to hydrophobic matching with fluid C(13:0) lipid chains. The stoichiometry ( N b = 23 lipids/monomer) of lipid-protein interaction from EPR demonstrates that FomA is not trimeric in membranes of diC(14:0) phosphatidylcholine and is consistent with a monomeric beta-barrel of 14-16 strands. The pronounced selectivity of interaction found with anionic spin-labeled lipids places basic residues of the protein in the vicinity of the polar-apolar membrane interfaces, consistent with current topology models. Comparison with similar data from the 8- to 22-stranded E. coli outer membrane proteins, OmpA, OmpG, and FhuA, supports the above conclusions.     

Effect of chain length and unsaturation on elasticity of lipid bilayers

Micropipette pressurization of giant bilayer vesicles was used to measure both elastic bending k(c) and area stretch K(A) moduli of fluid-phase phosphatidylcholine (PC) membranes. Twelve diacyl PCs were chosen: eight with two 18 carbon chains and degrees of unsaturation from one double bond (C18:1/0, C18:0/1) to six double bonds per lipid (diC18:3), two with short saturated carbon chains (diC13:0, diC14:0), and two with long unsaturated carbon chains (diC20:4, diC22:1). Bending moduli were derived from measurements of apparent expansion in vesicle surface area under very low tensions (0.001-0.5 mN/m), which is dominated by smoothing of thermal bending undulations. Area stretch moduli were obtained from measurements of vesicle surface expansion under high tensions (>0.5 mN/m), which involve an increase in area per molecule and a small-but important-contribution from smoothing of residual thermal undulations. The direct stretch moduli varied little (< +/-10%) with either chain unsaturation or length about a mean of 243 mN/m. On the other hand, the bending moduli of saturated/monounsaturated chain PCs increased progressively with chain length from 0.56 x 10(-19) J for diC13:0 to 1.2 x 10(-19) J for diC22:1. However, quite unexpectedly for longer chains, the bending moduli dropped precipitously to approximately 0.4 x 10(-19) J when two or more cis double bonds were present in a chain (C18:0/2, diC18:2, diC18:3, diC20:4). Given nearly constant area stretch moduli, the variations in bending rigidity with chain length and polyunsaturation implied significant variations in thickness. To test this hypothesis, peak-to-peak headgroup thicknesses h(pp) of bilayers were obtained from x-ray diffraction of multibilayer arrays at controlled relative humidities. For saturated/monounsaturated chain bilayers, the distances h(pp) increased smoothly from diC13:0 to diC22:1 as expected. Moreover, the distances and elastic properties correlated well with a polymer brush model of the bilayer that specifies that the elastic ratio (k(c)/K(A))(1/2) = (h(pp) - h(o))/24, where h(o) approximately 1 nm accounts for separation of the headgroup peaks from the deformable hydrocarbon region. However, the elastic ratios and thicknesses for diC18:2, diC18:3, and diC20:4 fell into a distinct group below the correlation, which showed that poly-cis unsaturated chain bilayers are thinner and more flexible than saturated/monounsaturated chain bilayers.     

Association of spin-labeled lipids with beta-barrel proteins from the outer membrane of Escherichia coli

The interaction of spin-labeled lipids with beta-barrel transmembrane proteins has been studied by the electron spin resonance (ESR) methods developed for alpha-helical integral proteins. The outer membrane protein OmpA and the ferrichrome-iron receptor FhuA from the outer membrane of Escherichia coli were reconstituted in bilayers of dimyristoylphosphatidylglycerol. The ESR spectra from phosphatidylglycerol spin labeled on the 14-C atom of the sn-2 chain contain a second component from motionally restricted lipids contacting the intramembranous surface of the beta-barrel, in addition to that from the fluid bilayer lipids. The stoichiometry of motionally restricted lipids, 11 and 32 lipids/monomer for OmpA and FhuA, respectively, is constant irrespective of the total lipid/protein ratio. It is proportional to the number of transmembrane beta-strands, eight for OmpA and 22 for FhuA, and correlates reasonably well with the intramembranous perimeter of the protein. Spin-labeled lipids with different polar headgroups display a differential selectivity of interaction with the two proteins. The more pronounced pattern of lipid selectivity for FhuA than for OmpA correlates with the preponderance of positively charged residues facing the lipids in the extensions of the beta-sheet and shorter interconnecting loops on the extracellular side of FhuA.     

Infrared dichroism of twisted beta-sheet barrels. The structure of E. coli outer membrane proteins

The infrared dichroic ratios of the amide bands from oriented beta-barrels yield an experimental value for the mean orientation, beta, of the beta-strands, relative to the barrel axis. For a barrel of n strands, this then gives the shear number, S, that characterizes the stagger of the beta-sheet. Combining values of beta and n specifies the barrel geometry by using the optimized model of Murzin, Lesk & Chothia for regular barrels. Application to published infrared data on the Escherichia coli outer membrane protein, OmpA yields S=9-10 (n=8), a barrel radius of 0.81(+/-0.01) nm, and an internal free volume of 0.031 nm(3) per residue, where the average twist of the beta-sheets is theta approximately 28 degrees, and their coiling angle is epsilon approximately 1 degrees. Hydrophobic matching of the 2.6 nm transmembrane stretch partly determines the shear number of the OmpA beta-barrel.     

The major outer membrane protein of Fusobacterium nucleatum (FomA) folds and inserts into lipid bilayers via parallel folding pathways

Membrane protein insertion and folding was studied for the major outer membrane protein of Fusobacterium nucleatum (FomA), which is a voltage-dependent general diffusion porin. The transmembrane domain of FomA forms a beta-barrel that is predicted to consist of 14 beta-strands. Here, unfolded FomA is shown to insert and fold spontaneously and quantitatively into phospholipid bilayers upon dilution of the denaturant urea, which was shown previously only for outer membrane protein A (OmpA) of Escherichia coli. Folding of FomA is demonstrated by circular dichroism and fluorescence spectroscopy, by SDS-polyacrylamide gel electrophoresis, and by single-channel recordings. Refolded FomA had a single-channel conductance of 1.1 nS at 1 M KCl, in agreement with the conductance of FomA isolated from membranes in native form. In contrast to OmpA, which forms a smaller eight-stranded beta-barrel domain, folding kinetics of the larger FomA were slower and provided evidence for parallel folding pathways of FomA into lipid bilayers. Two pathways were observed independent of membrane thickness with two different lipid bilayers, which were either composed of dicapryl phosphatidylcholine or dioleoyl phosphatidylcholine. This is the first observation of parallel membrane insertion and folding pathways of a beta-barrel membrane protein from an unfolded state in urea into lipid bilayers. The kinetics of both folding pathways depended on the chain length of the lipid and on temperature with estimated activation energies of 19 kJ/mol (dicapryl phosphatidylcholine) and 70 kJ/mol (dioleoyl phosphatidylcholine) for the faster pathways.     

Dehydration induces lateral expansion of polyunsaturated 18:0-22:6 phosphatidylcholine in a new lamellar phase.

To gain a better understanding of the biological role of polyunsaturated phospholipids, infrared (IR) linear dichroism, NMR, and x-ray diffraction studies have been conducted on the lyotropic phase behavior and bilayer dimensions of sn-1 chain perdeuterated 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (SDPC-d35), a mixed-chain saturated (18:0)-polyunsaturated (22:6 omega 3) lipid. SDPC films were hydrated at definite values of temperature (T) and relative humidity (RH). In excess water, the lipid forms exclusively lamellar phases in the temperature range 0--50 degrees C. Upon dehydration the lipid undergoes the main phase transition between the liquid-crystalline (L(alpha)) and gel (L(beta)) phase at T < 15 degrees C. Both the saturated and polyunsaturated chains adopt a stretched conformation in the L(beta) phase, presumably the all-trans (stearoyl) and angle iron or helical (docosahexaenoyl) one. A new fluid lamellar phase (L(alpha)') was found in partially hydrated samples at T > 15 degrees C. SDPC membranes expand laterally and contract vertically in the L(alpha)' phase when water was removed. This tendency is in sharp contrast to typical dehydration-induced changes of membrane dimensions. The slope of the phase transition lines in the RH-T phase diagram reveal that the lyotropic L(alpha)'-L(alpha) and L(beta)-L(alpha) transitions are driven by enthalpy and entropy, respectively The possible molecular origin of the phase transitions is discussed. The properties of SDPC are compared with that of membranes of monounsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC-d31).     

Lipid chain-length dependence for incorporation of alamethicin in membranes: electron paramagnetic resonance studies on TOAC-spin labeled analogs

Alamethicin is a 19-residue hydrophobic peptide, which is extended by a C-terminal phenylalaninol but lacks residues that might anchor the ends of the peptide at the lipid-water interface. Voltage-dependent ion channels formed by alamethicin depend strongly in their characteristics on chain length of the host lipid membranes. EPR spectroscopy is used to investigate the dependence on lipid chain length of the incorporation of spin-labeled alamethicin in phosphatidylcholine bilayer membranes. The spin-label amino acid TOAC is substituted at residue positions n = 1, 8, or 16 in the sequence of alamethicin F50/5 [TOAC(n), Glu(OMe)(7,18,19)]. Polarity-dependent isotropic hyperfine couplings of the three TOAC derivatives indicate that alamethicin assumes approximately the same location, relative to the membrane midplane, in fluid diC(N)PtdCho bilayers with chain lengths ranging from N = 10-18. Residue TOAC(8) is situated closest to the bilayer midplane, whereas TOAC(16) is located farther from the midplane in the hydrophobic core of the opposing lipid leaflet, and TOAC(1) remains in the lipid polar headgroup region. Orientational order parameters indicate that the tilt of alamethicin relative to the membrane normal is relatively small, even at high temperatures in the fluid phase, and increases rather slowly with decreasing chain length (from 13 degrees to 23 degrees for N = 18 and 10, respectively, at 75 degrees C). This is insufficient for alamethicin to achieve hydrophobic matching. Alamethicin differs in its mode of incorporation from other helical peptides for which transmembrane orientation has been determined as a function of lipid chain length.     

Formation of the antiplanar-antiplanar phosphate conformation of dilauroylphosphatidylcholine bilayers

Infrared spectroscopy was used to investigate lipid conformational changes that occur in dilauroylphosphatidylcholine (diC12PC) bilayers with and without fatty-acid-amino-acids as guest molecules in the membrane. Incorporating 2.5 mole% N-decanoylglycine (decgly) into diC12PC liposomes caused formation of the antiplanar-antiplanar (ap-ap) phosphodiester conformation which was stable in room temperature IR spectra. Several other fatty-acid-amino-acids incorporated into diC12PC bilayers were found to also elicit the ap-ap phosphodiester conformation. Unlike these diC12PC/fatty-acid-amino-acid mixed bilayers, pure diC12PC bilayers would form the ap-ap phosphodiester conformation only under low temperature incubation conditions. Dry diC12PC films incubated at 5 degrees C for 0.5 h (brief incubation) or 16 h (prolonged incubation), and then rapidly hydrated (i.e., vortexed at 25 degrees C in D2O), caused the ap-ap phosphodiester conformation to persist in the diC12PC liposomes equilibrated to room temperature. Slow hydration for 16 h at 5 degrees C in both buffered and non-buffered D2O of diC12PC lipid films also produced the ap-ap phosphodiester conformation. In contrast, slow hydration for 16 h at 5 degrees C in PBS/D2O of diC12PC/decgly mixed films caused the greatest number of ap-ap phosphodiester conformers. Using pure diC12PC bilayers, infrared data indicate that incubation of diC12PC films causes the headgroup phosphodiester conformation to change from gauche-gauche (g-g) conformation to the ap-ap conformation. Under all liposome formation conditions examined, no changes in hydration of either the phosphate group or the carbonyl ester group were detected and in addition, no trans/gauche conformational changes in the acyl chain were observed.     

Fourier transform infrared spectroscopy and site-directed isotope labeling as a probe of local secondary structure in the transmembrane domain of phospholamban.

Phospholamban is a 52-amino acid residue membrane protein that regulates Ca(2+)-ATPase activity in the sarcoplasmic reticulum of cardiac muscle cells. The hydrophobic C-terminal 28 amino acid fragment of phospholamban (hPLB) anchors the protein in the membrane and may form part of a Ca(2+)-selective ion channel. We have used polarized attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy along with site-directed isotope labeling to probe the local structure of hPLB. The frequency and dichroism of the amide I and II bands appearing at 1658 cm-1 and 1544 cm-1, respectively, show that dehydrated and hydrated hPLB reconstituted into dimyristoylphosphatidycholine bilayer membranes is predominantly alpha-helical and has a net transmembrane orientation. Specific local secondary structure of hPLB was probed by incorporating 13C at two positions in the protein backbone. A small band seen near 1614 cm-1 is assigned to the amide I mode of the 13C-labeled amide carbonyl group(s). The frequency and dichroism of this band indicate that residues 39 and 46 are alpha-helical, with an axial orientation that is approximately 30 degrees relative to the membrane normal. Upon exposure to 2H2O (D2O), 30% of the peptide amide groups in hPLB undergo a slow deuterium/hydrogen exchange. The remainder of the protein, including the peptide groups of Leu-39 and Leu-42, appear inaccessible to exchange, indicating that most of the hPLB fragment is embedded in the lipid bilayer. By extending spectroscopic characterization of PLB to include hydrated, deuterated as well as site-directed isotope-labeled hPLB films, our results strongly support models of PLB that predict the existence of an alpha-helical hydrophobic region spanning the membrane domain.     

Folding kinetics of the outer membrane proteins OmpA and FomA into phospholipid bilayers

The folding mechanism of outer membrane proteins (OMPs) of Gram-negative bacteria into lipid bilayers has been studied using OmpA of E. coli and FomA of F. nucleatum as examples. Both, OmpA and FomA are soluble in unfolded form in urea and insert and fold into phospholipid bilayers upon strong dilution of the denaturant urea. OmpA is a structural protein and forms a small ion channel, composed of an 8-stranded transmembrane beta-barrel domain. FomA is a voltage-dependent porin, predicted to form a 14 stranded beta-barrel. Both OMPs fold into a range of model membranes of very different phospholipid compositions. Three membrane-bound folding intermediates of OmpA were discovered in folding studies with dioleoylphosphatidylcholine bilayers that demonstrated a highly synchronized mechanism of secondary and tertiary structure formation of beta-barrel membrane proteins. A study on FomA folding into lipid bilayers indicated the presence of parallel folding pathways for OMPs with larger transmembrane beta-barrels.     

Bacteriorhodopsin: the effect of bilayer thickness on 2D-array formation, and the structural re-alignment of retinal through the photocycle

From our earlier extensive protein-lipid reconstitution studies, the conditions under which bacteriorhodopsin forms organised 2D arrays in large unilamellar vesicles have been established using freeze-fracture electron microscopy. In a background bilayer matrix of phosphatidylcholine (diC(14:0)), the protein can form arrays only when the anionic purple membrane lipid, phosphatidylglycerol phosphate (or the sulphate derivative) is present. Here we have now extended this work to investigate the effect of bilayer thickness on array formation. Phosphatidylcholines with various chain lengths (diC(12:0), diC(14:0) and diC(16:0)) and which form bilayers of well defined bilayer thickness, have been used as the matrix into which bacteriorhodopsin, together with minimal levels (c. 4-10 lipids per bacteriorhodopsin) of diphytanyl phosphatidyl-glycerol phosphate, has been reconstituted. Arrays are formed in all complexes and bhickness appears only to alter the type of array formed, either as an orthogonal or as an hexagonal array. Secondly, we have previously deduced the entire conformation of retinal within the bacteriorhodopsin binding pocket in oriented purple membrane fragments. Using solid state deuterium NMR of the specifically deutero-methylated retinal labelled at each of the methyl positions in the molecule, the C-CD(3) bond vectors of the chromophore have been resolved to +/- 2 degrees . The ring conformation is 6-S-trans, but the polyene chain is slightly curved when in the protein binding site. Here, we describe studies on the protein in both the ground state and the trapped M(412)-state of the photocycle, to show that the orientation of the central methyl group (C(19)) on the polyene chain, which is at 40 degrees +/- 1 degrees with respect to the membrane normal, only changes its orientation by approximately 4 degrees upon 13-cis-isomerization. Thus, it is the Schiff base end of the chromophore which moves upon light incidence acting as a local switch on the protein in the photocycle, whilst the ring end of the chromophore moves rather less.     

Water permeability and mechanical strength of polyunsaturated lipid bilayers

Micropipette aspiration was used to test mechanical strength and water permeability of giant-fluid bilayer vesicles composed of polyunsaturated phosphatidylcholine PC lipids. Eight synthetic-diacyl PCs were chosen with 18 carbon chains and degrees of unsaturation that ranged from one double bond (C18:0/1, C18:1/0) to six double bonds per PC molecule (diC18:3). Produced by increasing pipette pressurization, membrane tensions for lysis of single vesicles at 21 degrees C ranged from approximately 9 to 10 mN/m for mono- and dimono-unsaturated PCs (18:0/1, 18:1/0, and diC18:1) but dropped abruptly to approximately 5 mN/m when one or both PC chains contained two cis-double bonds (C18:0/2 and diC18:2) and even lower approximately 3 mN/m for diC18:3. Driven by osmotic filtration following transfer of individual vesicles to a hypertonic environment, the apparent coefficient for water permeability at 21 degrees C varied modestly in a range from approximately 30 to 40 microm/s for mono- and dimono-unsaturated PCs. However, with two or more cis-double bonds in a chain, the apparent permeability rose to approximately 50 microm/s for C18:0/2, then strikingly to approximately 90 microm/s for diC18:2 and approximately 150 microm/s for diC18:3. The measurements of water permeability were found to scale exponentially with the reduced temperatures reported for these lipids in the literature. The correlation supports the concept that increase in free volume acquired in thermal expansion above the main gel-liquid crystal transition of a bilayer is a major factor in water transport. Taken together, the prominent changes in lysis tension and water permeability indicate that major changes occur in chain packing and cohesive interactions when two or more cis-double bonds alternate with saturated bonds along a chain.     

Infrared dichroism from the X-ray structure of bacteriorhodopsin

A detailed comparison with the three-dimensional protein structure provides a stringent test of the models and parameters commonly used in determining the orientation of the alpha-helices from the linear dichroism of the infrared amide bands, particularly in membranes. The order parameters of the amide vibrational transition moments are calculated for the transmembrane alpha-helices of bacteriorhodopsin by using the crystal structure determined at a resolution of 1.55 A (PDB accession number 1C3W). The dependence on the angle delta(M) that the transition moment makes with the peptide carbonyl bond is fit by the expression ((3)/(2)S(alpha) cos(2) alpha)cos(2)(delta(M) + beta) - 1/2S(alpha), where S(alpha) (0.91) is the order parameter of the alpha-helices, alpha (13 degrees ) is the angle that the peptide plane makes with the helix axis, and beta (11 degrees ) is the angle that the peptide carbonyl bond makes with the projection of the helix axis on the peptide plane. This result is fully consistent with the model of nested axial distributions commonly used in interpreting infrared linear dichroism of proteins. Comparison with experimental infrared dichroic ratios for bacteriorhodopsin yields values of Theta(A) = 33 +/- 1 degree, Theta(I) = 39.5 +/- 1 degree, and Theta(II) = 70 +/- 2 degrees for the orientation of the transition moments of the amide A, amide I, and amide II bands, respectively, relative to the helix axis. These estimates are close to those found for model alpha-helical polypeptides, indicating that side-chain heterogeneity and slight helix imperfections are unlikely to affect the reliability of infrared measurements of helix orientations.     

Conversion of the FhuA transport protein into a diffusion channel through the outer membrane of Escherichia coli.

The FhuA receptor protein is involved in energy-coupled transport of Fe3+ via ferrichrome through the outer membrane of Escherichia coli. Since no energy source is known in the outer membrane it is assumed that energy is provided through the action of the TonB, ExbB and ExbD proteins, which are anchored to the cytoplasmic membrane. By deleting 34 amino acid residues of a putative cell surface exposed loop, FhuA was converted from a ligand specific transport protein into a TonB independent and nonspecific diffusion channel. The FhuA deletion derivative FhuA delta 322-355 formed stable channels in black lipid membranes, in contrast to wild-type FhuA which did not increase membrane conductance. The single-channel conductance of the FhuA mutant channels was at least three times larger than that of the general diffusion porins of E. coli outer membrane. It is proposed that the basic structure of FhuA in the outer membrane is a channel formed by beta-barrels. Since the loop extending from residue 316 to 356 is part of the active site of FhuA, it probably controls the permeability of the channel. The transport-active conformation of FhuA is mediated by a TonB-induced conformational change in response to the energized cytoplasmic membrane. The ferrichrome transport rate into cells expressing FhuA delta 322-355 increased linearly with increasing substrate concentration (from 0.5 to 20 microM), in contrast to FhuA wild-type cells, which displayed saturation at 5 microM. This implies that in wild-type cells ferrichrome transport through the outer membrane is the rate-limiting step and that TonB, ExbB and ExbD are only required for outer membrane transport.     

Preparation of the FhuA (TonA) receptor protein from cell envelopes of an overproducing strain of Escherichia coli K-12.

A rapid and simple method for purification of the FhuA receptor protein from cell envelopes of a FhuA-overproducing strain of Escherichia coli K-12 was developed. The overproduction of FhuA was programmed by the thermoamplifiable plasmid pHK232, which carried the fhuACD genes of pLC19-19 of the Clarke and Carbon collection. At low temperature (27 degrees C), pHK232 specified the overproduction of FhuA to levels comparable to those of major outer membrane proteins OmpF, OmpC, and OmpA. The amount of these proteins in the outer membrane was reduced along with overproduction of FhuA. Upon runaway replication of pHK232 at 37 degrees C, the precursor of the FhuA protein, proFhuA, was also accumulated in the cell envelope in amounts similar to FhuA. For extraction of the FhuA protein, crude cell envelopes were washed with 2% Triton X-100-6 M urea to remove less tightly bound proteins. Then FhuA but not proFhuA was solubilized by treating Triton X-100-urea-washed membranes with 1% octylglucoside-1 mM EDTA. This procedure yielded FhuA protein free from other membrane proteins. The amount of lipopolysaccharide and phospholipids was low and ranged from 5 to 15% and 10 to 25% of the weight of the FhuA protein, respectively. As shown by direct inactivation and by competition assays, the isolated FhuA protein retained receptor activity for ferrichrome, albomycin, colicin M, and phages T5 and T1.     

Two types of hydrocarbon chain interdigitation in sphingomyelin bilayers

Vibrational Raman spectroscopic experiments have been performed as a function of temperature on aqueous dispersions of synthetic DL-erythro-N-lignoceroylsphingosylphosphocholine [C(24):SPM], a racemic mixture of two highly asymmetric hydrocarbon chain length sphingomyelins. Raman spectral peak-height intensity ratios of vibrational transitions in the C-H stretching-mode region show that the C(24):SPM-H2O system undergoes two thermal phase transitions centered at 48.5 and 54.5 degrees C. Vibrational data for fully hydrated C(24):SPM are compared to those of highly asymmetric phosphatidylcholine dispersions. The Raman data are consistent with the plausible model that the lower temperature transition can be ascribed to the conversion of a mixed interdigitated gel state (gel II) to a partially interdigitated gel state (gel I) and that the higher temperature transition corresponds to a gel I----liquid-crystalline phase transition. The observation of a mixed interdigitated gel state (gel II) at temperatures below 48.5 degrees C implies that biological membranes may have lipid domains in which some of the lipid hydrocarbon chains penetrate completely across the entire hydrocarbon width of the lipid bilayer.     

Tilt, twist, and coiling in beta-barrel membrane proteins: relation to infrared dichroism

The x-ray coordinates of beta-barrel transmembrane proteins from the porins superfamily and relatives are used to calculate the mean tilt of the beta-strands and their mean local twist and coiling angles. The 13 proteins examined correspond to beta-barrels with 8 to 22 strands, and shear numbers ranging from 8 to 24. The results are compared with predictions from the model of Murzin, Lesk, and Chothia for symmetrical regular barrels. Good agreement is found for the mean strand tilt, but the twist angles are smaller than those for open beta-sheets and beta-barrels with shorter strands. The model is reparameterised to account for the reduced twist characteristic of long-stranded transmembrane beta-barrels. This produces predictions of both twist and coiling angles that are in agreement with the mean values obtained from the x-ray structures. With the optimized parameters, the model can then be used to determine twist and coiling angles of transmembrane beta-barrels from measurements of the amide band infrared dichroism in oriented membranes. Satisfactory agreement is obtained for OmpF. The strand tilt obtained from the x-ray coordinates, or from the reparameterised model, can be combined with infrared dichroism measurements to obtain information on the orientation of the beta-barrel assembly in the membrane.     

Influence of N-dodecyl-N,N-dimethylamine N-oxide on the activity of sarcoplasmic reticulum Ca(2+)-transporting ATPase reconstituted into diacylphosphatidylcholine vesicles: efects of bilayer physical parameters

Sarcoplasmic reticulum Ca-transporting ATPase (EC 3.6.1.38) was isolated from rabbit white muscle, purified and reconstituted into vesicles of synthetic diacylphosphatidylcholines with monounsaturated acyl chains using the cholate dilution method. In fluid bilayers at 37 degrees C, the specific activity of ATPase displays a maximum (31.5+/-0.8 IU/mg) for dioleoylphosphatidylcholine (diC18:1PC) and decreases progressively for both shorter and longer acyl chain lengths. Besides the hydrophobic mismatch between protein and lipid bilayer, changes in the bilayer hydration and lateral interactions detected by small angle neutron scattering (SANS) can contribute to this acyl chain length dependence. When reconstituted into dierucoylphosphatidylcholine (diC22:1PC), the zwitterionic surfactant N-dodecyl-N,N-dimethylamine N-oxide (C12NO) stimulates the ATPase activity from 14.2+/-0.6 to 32.5+/-0.8 IU/mg in the range of molar ratios C12NO:diC22:1PC=0/1.2. In dilauroylphosphatidylcholines (diC12:0PC) and diC18:1PC, the effect of C12NO is twofold-the ATPase activity is stimulated at low and inhibited at high C12NO concentrations. In diC18:1PC, it is observed an increase of activity induced by C12NO in the range of molar ratios C12NO:diC18:1PC< or =1.3 in bilayers, where the bilayer thickness estimated by SANS decreases by 0.4+/-0.1 nm. In this range, the 31P-NMR chemical shift anisotropy increases indicating an effect of C12NO on the orientation of the phosphatidylcholine dipole N(+)-P- accompanied by a variation of the local membrane dipole potential. A decrease of the ATPase activity is observed in the range of molar ratios C12NO:diC18:1PC=1.3/2.5, where mixed tubular micelles are detected by SANS in C12NO+diC18:1PC mixtures. It is concluded that besides hydrophobic thickness changes, the changes in dipole potential and curvature frustration of the bilayer could contribute as well to C12NO effects on Ca(2+)-ATPase activity.     

Infrared dichroism of isotope-edited alpha-helices and beta-sheets

Isotope editing of amide infrared bands not only localises secondary structural elements within the protein but also yields conformational information that is not available from the linear dichroism of aligned samples without isotope editing. The additional information that can be derived on the orientational distribution of alpha-helices in membranes by the combined use of different amide bands and several positions of labelling is presented here. Also, the relationship between the azimuthal orientation of the transition moment and the protein structure is treated explicitly. A comprehensive analysis of the infrared dichroism for beta-sheets and beta-barrels is given here, for the first time. The orientation of the individual transition moments in a beta-sheet that is essential for this analysis is derived for the different amide bands.