Cation transport properties of a synthetic Ca2+-selective peptide ionophore in phospholipid and sarcoplasmic reticulum vesicles.

Transport by the synthetic cyclic peptide ionophore CYCLEX-2E (Deber, C.M. Young, M.E.M., and Tom-Kun, J. (1980) Biochemistry 19, 6194-6198), which in contrast to Ca2+ ionophore A23187 contains no ionizable protons, has been studied with respect to Ca2+ and Na+ transport, and the involvement of exchanged, or counter-transported ions during the transport process. CYCLEX-2E was found to equilibrate Na+ and Ca2+ gradients across phospholipid vesicle membranes. Experiments using the indicator dye Arsenazo III established that calcium ions were indeed reaching the aqueous intravesicular compartments. Absence of metal cations in the external buffer slowed, but did not eliminate, the efflux of Ca2+ from phosphatidylcholine vesicles. As an example of its activity in a biological membrane, CYCLEX-2E was shown to be capable of producing Ca2+ efflux from sarcoplasmic reticulum vesicles which has been loaded with Ca2+ in an ATP-dependent manner. The overall results suggest that in transport by synthetic peptide ionophores typified by CYCLEX-2E, electroneutrality is achieved either through (a) peptide-mediated compensating (but not coupled) fluxes of other cations, or where this is not an option, by (b) transmembrane diffusion of permeant ions such as H+, OH-, or Cl-.

Bilayer-stabilizing properties of myelin basic protein in dioleoylphosphatidylethanolamine systems.

31P-NMR and X-ray diffraction techniques are used to study the comparative ability of myelin basic protein (MBP) vs. other basic proteins to convert hexagonal (HII) phases to stable lamellar (L alpha) structures. Pure dioleoylphosphatidylethanolamine (DOPE) at pH 9 and 7, and mixtures of DOPE/phosphatidylserine (PS) (95:5 and 80:20% w/w) at pH 7 were employed for this investigation. The polymorphic behavior of the lipid suspensions was evaluated in the presence and absence of several basic proteins (MBP, calf thymus histone, lysozyme, melittin) and the cationic polypeptide, polylysine (PL). Each of the proteins and PL was capable of binding the pure DOPE HII phase at pH 9 but with varying morphological consequences, i.e., lamellar stabilization (MBP, histone, PL), formation of new protein-DOPE HII phases (lysozyme) or lipid disordering/vesiculation (melittin). Reduction to pH 7 resulted in the dissociation of protein from DOPE - with the exception of melittin - and the reformation of a pure lipid HII phase. Additions of PS to DOPE at pH 7 facilitated protein binding, but among the proteins examined, only MBP was capable of converting the lipid suspension into a stable multilamellar form. Differences in the lipid morphology produced by each protein are discussed in terms of protein physicochemical characteristics. In addition, a possible relationship between MBP-lipid interactions and the stability of myelin sheath lipid multilayers is inferred from the significant bilayer-stabilizing capacity of MBP.

Spontaneous vesicularization of myelin lipids is counteracted by myelin basic protein.

Hand-vortexed dispersions of several lipids (cerebrosides, sulfatides, PC, PE, PS and sphingomyelin), mixed in the ratios found for these categories of lipids in myelin, exhibit 31P-NMR spectra which have contributions from both isotropic and lamellar resonances. Investigation of this system by freeze-fracture electron microscopy and X-ray diffraction revealed that this lipid mixture has spontaneously formed small unilamellar vesicles (SUVs) (diam. approximately 400 A) and large highly convoluted unilamellar vesicles (LUVs) (diam. approximately 1000 A), the latter possibly resulting from aggregation and fusion of the SUV structures. This vesicularization of the myelin lipids was reversed by the addition of myelin basic protein: only large multilamellar aggregates were formed in the presence of protein, as shown by all three experimental methods. Although no rigorous physical-chemical explanation for these phenomena is yet available, the possibility is suggested that the high concentration of cerebrosides and/or phosphatidylethanolamine in this particular mixture of myelin lipids play pivotal roles in the formation of these unusual vesicles. Spontaneous vesicularization of myelin lipids is discussed as a potential pathway toward destabilization of the myelin sheath.

Non-random distribution of amino acids in the transmembrane segments of human type I single span membrane proteins.

The distribution of amino acids in the transmembrane segments and flanking regions of 115 human type I single span (amino terminus extracellular and carboxyl terminus cytosolic) plasma membrane proteins was found to be non-random. In this sample, Ile was preferentially localized to the amino-terminal region of the hydrophobic transmembrane segments, followed by Val, while Leu predominated in the carboxyl-terminal half of the segment. Although Gly residues were preferentially located in the transmembrane segment, this residue was excluded from the carboxyl-terminal and adjacent boundary regions. Aromatic residues (Tyr, Trp and Phe) occurred preferentially at the cytoplasmic boundary, with Trp also favored at the extracellular boundary. The extracellular flanking sequence amino-terminal to the transmembrane segment was enriched in residues predicted to initiate helix formation (Pro, Asn and Ser), while Arg and Lys were enriched in the cytoplasmic flank where they may function as topological determinants. The positional preferences of these particular amino acids within the transmembrane segment and flanking regions suggests that, in addition to lipid-protein interactions, these residues may participate in specific protein-protein interactions. A consensus sequence motif for type I membrane proteins is proposed and its role in the biosynthesis, folding, assembly and function of these segments is discussed.

Transmembrane aromatic amino acid distribution in P-glycoprotein. A functional role in broad substrate specificity.

Multidrug resistance (MDR) in cancer cells is associated with overexpression of P-glycoprotein (Pgp), a membrane protein which interacts with structurally diverse hydrophobic molecules of high membrane affinity. In an analysis of the molecular basis for this broad range of substrate specificity, we found that the transmembrane (TM) regions of Pgp are rich in highly conserved aromatic amino acid residues. Computer-generated three-dimensional model structures showed that a typical substrate, rhodamine 123, can intercalate between three to four phenylalanine side-chains in any of several Pgp TM helices with minimal protrusion of the drug into bulk lipid, and that five to six (of the 12 Pgp putative TM segments) helices can facilitate transport through creation of a sterically compatible pore. In contrast to the case for proteins involved in the transport of membrane-impermeable, relatively polar substrates, the "transport path" for Pgp substrates need not be polar, and may involve either an internal channel occupied largely by aromatic side-chains, or external gaps along TM helix-lipid interfaces. Weakly polar interactions between drug cationic sites and Pgp aromatic residues contribute additionally to overall protein/drug binding. The ability of Pgp to recognize and efflux structurally diverse molecules suggests that rather than a unique structure, the Pgp channel may maintain the intrinsic capacity to undergo wide-ranging drug-dependent dynamic reorganization.

Packing of coat protein amphipathic and transmembrane helices in filamentous bacteriophage M13: role of small residues in protein oligomerization.

Filamentous bacteriophage M13, an important cloning and phage display vector, is encapsulated by ca 2700 copies of its 50-residue major coat protein (gene 8). This protein occurs as a membrane protein while stably inserted into its E. coli host inner membrane, and as a coat protein upon assembly and packing onto phage DNA in the lipid-free virion. To examine the specific protein-protein interactions underlying these processes, we used a combination of randomized and saturation mutagenesis of the entire gene 8 to assess the susceptibility of each position to mutation. In the resulting library of ca 100 viable M13 mutants, "small" residues (Ala,Gly,Ser), which constitute the non-polar face of the N-terminal amphipathic helical segment, and a face of the hydrophobic (effective transmembrane) helical segment, were found to be highly conserved. These results support a model in which coat protein packing is stabilized by the presence within each protein subunit of two "oligomerization segments", i.e. specific helical regions with faces rich in small residues which function to promote the close approach of alpha-helices.

TM Finder: a prediction program for transmembrane protein segments using a combination of hydrophobicity and nonpolar phase helicity scales.

Based on the principle of dual prediction by segment hydrophobicity and nonpolar phase helicity, in concert with imposed threshold values of these two parameters, we developed the automated prediction program TM Finder that can successfully locate most transmembrane (TM) segments in proteins. The program uses the results of experiments on a series of host-guest TM segment mimic peptides of prototypic sequence KK AAAXAAAAAXAAWAAXAAAKKKK-amide (where X = each of the 20 commonly occurring amino acids) through which an HPLC-derived hydropathy scale, a hydrophobicity threshold for spontaneous membrane insertion, and a nonpolar phase helical propensity scale were determined. Using these scales, the optimized prediction algorithm of TM Finder defines TM segments by first searching for competent core segments using the combination of hydrophobicity and helicity scales, and then performs a gap-joining operation, which minimizes prediction bias caused by local hydrophilic residues and/or the choice of window size. In addition, the hydrophobicity threshold requirement enables TM Finder to distinguish reliably between membrane proteins and globular proteins, thereby adding an important dimension to the program. A full web version of the TM Finder program can be accessed at http://www.bioinformatics-canada.org/TM/.

Glycine and beta-branched residues support and modulate peptide helicity in membrane environments.

Transmembrane (TM) segments of integral membrane proteins are putatively alpha-helical in conformation once inserted into the membrane, yet consist of primary sequences rich in residues known in soluble proteins as helix-breakers (Gly) and beta-sheet promoters (Ile, Val, Thr). To examine the specific 2 degrees structure propensities of such residues in membrane environments, we have designed and synthesized a series of 20-residue peptides with 'guest' hydrophobic segments--expected to provide three turns of incipient alpha-helix content--embedded in 'host' hydrophilic (Lys-Ser) matrices. Circular dichroism (CD) spectra of the model peptides in water showed that significant helical content was observed only for peptides with high Ala content; others behaved as 'random coils'. However, in the membrane-mimetic environment of sodium dodecylsulfate (SDS) micelles, it was found that Gly can be accommodated as readily as Ala, and Ile or Val as readily as Leu, in hydrophobic alpha-helices. Further subtleties of structural preferences could be observed in electrically-neutral lyso-phosphatidylcholine (LPC) micelles, where helical propensity decreased in the order Ala-Leu-rich > Gly-Leu-rich > Gly-Ile(Val)-rich hydrophobic segments. The results conjure a role of environment-dependent helix-modulation for Gly, Ile, and Val residues--and suggest that these residues may provide, in part, the structural basis for conformational transitions within or adjacent to membrane domains, such as those accompanying membrane insertion and/or required for transport or signalling functions.

Cystic fibrosis transmembrane conductance regulator: expression and helicity of a double membrane-spanning segment.

The gene responsible for cystic fibrosis encodes a membrane protein--the 1480-residue cystic fibrosis transmembrane conductance regulator (CFTR)--in which membrane-based CF-phenotypic mutants alter pore structure and/or impair ion transport. We report the preparation in milligram quantities and conformational characterization of a polypeptide comprised of CFTR transmembrane (TM) segments 3-4, a putative 'helical hairpin' portion of the CFTR TM1-6 domain. The TM segment 3-4 of CFTR was expressed in E. coli as a fusion protein linked to the C-terminus of His-tagged thioredoxin. Nickel chelate affinity chromatography, followed by release from the carrier by digestion with thrombin protease, gave free CFTR(TM3-4). Monitoring of the folding properties and conformational state(s) of the TM3-4 polypeptide using circular dichroism spectroscopy indicated a partial alpha-helical conformation in aqueous buffer, with up to 30% increase in alpha-helical content observed in membrane-mimetic environments.

Central nervous system myelin: structure, function, and pathology.

Multiple sclerosis (MS) and a number of related distinctive diseases are characterized by the active degradation of central nervous system (CNS) myelin, an axonal sheath comprised essentially of proteins and lipids. These demyelinating diseases appear to arise from complex interactions of genetic, immunological, infective, and biochemical mechanisms. While circumstances of MS etiology remain hypothetical, one persistent theme involves recognition by the immune system of myelin-specific antigens derived from myelin basic protein (MBP), the most abundant extrinsic myelin membrane protein, and/or another equally susceptible myelin protein or lipid component. Knowledge of the biochemical and physical-chemical properties of myelin proteins and lipids, particularly their composition, organization, structure, and accessibility with respect to the compacted myelin multilayers, thus becomes central to the understanding of how and why these antigens become selected during the development of MS. This review focuses on current understanding of the molecular basis underlying demyelinating disease as it may relate to the impact of the various protein and lipid components on myelin morphology; the precise molecular architecture of this membrane as dictated by protein-lipid and lipid-lipid interactions; and the relationship, if any, between the protein/lipid components and the destruction of myelin in pathological situations.

Gas chromatographic/mass spectrometric analysis of methylphenidate (ritalin) in serum.

A gas chromatographic/mass spectrometric (GG/MS) procedure for the determination of methylphenidate in 1 mL of serum or plasma is reported employing ethylphenidate as internal standard. A 50/50 (V/V) mixture of benzene and hexane is used to extract the methylphenidate and ethylphenidate from plasma. After evaporation of solvent the residue is redissolved in 50 microL hexane. Methyl- and ethylphenidate are then derivatized by the addition of 50 ML of trifluoroacetic anhydride and the TFA derivatives are injected into a quadrupole GC/MS for analysis. This method has a lower limit of sensitivity for methylphenidate of 2.0 micrograms/L. The between-day precision study yielded coefficients of variation of 10.4% and 14.8% at methylphenidate concentrations of 25.6 and 5.2 micrograms/L respectively. The assay has been used to investigate the pharmacokinetics of methylphenidate administered to 6 children for treatment of hyperkinesis. The study yielded ranges for volume of distribution, elimination half-life, and total body clearance of 5.0-6.8 L/kg, 1.6-2.7 hours, and 1.4-2.9 L/kg-hr respectively.

The modulation of bovine milk D-galactosyltransferase by various phosphatidylethanolamines.

To investigate the possible role of nonbilayer phases in the modulation of glycosyltransferase activity, bovine milk D-galactosyltransferase has been studied in phosphatidylethanolamine (PE) membranes, including soybean PE, egg PE, PE prepared by transphosphatidylation of egg PC, bovine brain PE, plasmalogen PE, and DPPE. The gel-to-liquid crystalline transition (TC) and the lamellar-to-hexagonal transitions (TH) are known for most of the PE compounds. The lower the TC (or TH) value, the greater the stimulation of galactosyltransferase activity in both the lactose- and N-acetyllactosamine-synthetase reactions. No correlation was found between either TC or TH value and the break in the Arrhenius plots for the N-acetyllactosamine synthetase. In membranes consisting of mixtures of PE with PC, the dominant effect was that of PC. The stimulation of activity in the mixed-lipid systems was never greater than that produced by PC alone, therefore the enzyme showed a definite preference for PC in the mixtures.

Alginate as an auxiliary bacterial membrane: binding of membrane-active peptides by polysaccharides.

The chronicity of Pseudomonas aeruginosa infections in cystic fibrosis (CF) patients is characterized by overproduction of the exopolysaccharide alginate, in which biofilm bacteria are embedded. Alginate apparently contributes to the antibiotic resistance of bacteria in this form by acting as a diffusion barrier to positively charged antimicrobial agents. We have been investigating cationic antimicrobial peptides (CAPs) (prototypic sequence: KKAAAXAAAAAXAAWAAXAAAKKKK-NH(2), where X is any of the 20 commonly occurring amino acids) that were originally designed as transmembrane mimetic peptides. Peptides of this group above a specific hydrophobicity threshold insert spontaneously into membranes and have antibacterial activity at micromolar concentrations. While investigating the molecular basis of biofilm resistance to peptides, we found that the anionic alginate polysaccharide induces conformational changes in the most hydrophobic of these peptides typically associated with insertion of such peptides into membrane environments [Chan et al., J. Biol. Chem. (2004) vol. 279, pp. 38749-38754]. Through a combination of experiments measuring release of the fluorescent dye calcein from phospholipid vesicles, peptide interactions with vesicles in the presence and absence of alginate, and affinity of peptides for alginate as a function of net peptide core hydrophobicity, we show here that alginate offers a microenvironment that provides a protective mechanism for the encased bacteria by both binding and promoting the self-association of the CAPs. The overall results indicate that hydrophilic alginate polymers contain a significant hydrophobic compartment, and behave as an 'auxiliary membrane' for bacteria, thus identifying a unique protective role for biofilm exopolysaccharide matrices.

Peptide models for protein-mediated cation transport.

Substances which can perturb the transmembrane cation balance in a predictable manner have wide-ranging uses in the study of cellular processes. We have undertaken to examine transmembrane calcium transport on the molecular level through the design and synthesis of a series of ionophoric peptides as models for protein-mediated calcium transport. General mechanisms for carrier-mediated membrane transport are discussed. Cation transport profiles are presented for transport by synthetic peptides of structure cyclo(Glu(OR)-Sar-Gly-(N-R1)-Gly)2, where R = benzyl ester or H; R1 = n-decyl or cyclohexyl. Transport of physiologically abundant cations across "liquid membranes" in Pressman cells mediated by cyclo(Glu-Sar-Gly-(N-decyl)Gly)2 was observed to be essentially calcium specific, as long as calcium ions were present in the system. Multilamellar and unilamellar phosphatidylcholine vesicles were each found to be emptied of internal 45Ca2+ ions upon addition of cyclo(Glu(OBz)-Sar-Gly-(N-cyclohexyl)Gly)2 to the vesicle suspension. The results are compared with the naturally occurring calcium ionophore A23187.

D-glucose binding increases secondary structure of human erythrocyte monosaccharide transport protein.

Purified hexose transport protein ("band 4.5") from human erythrocytes, reconstituted in vesicles of its endogenous lipids, displays minima in its circular dichroism (CD) spectrum at 222 and 207 nm, a pattern diagnostic for alpha-helical content of proteins. Upon addition of D-glucose, a saturable increment of +10-12% in negative ellipticity at 222 nm is observed stereospecifically and reproducibly. Addition of L-glucose had no effect on the CD spectrum of the transport protein. Addition of cytochalasin B (CB), a reversible inhibitor of hexose transport, had no effect itself on transporter CD spectra, but restored the spectrum at 222 nm to its original value when added in the presence of D-glucose. The observed D-glucose-induced increase in ordered secondary structure is proposed to result from incorporation into the membrane of a segment of the transport protein originally at a membrane-water interface.

A lipid vesicle system for probing voltage-dependent peptide-lipid interactions: application to alamethicin channel formation.

A membrane potential is shown to be established in phosphatidylcholine/cholesterol unilamellar vesicles using valinomycin in conjunction with a potassium ion gradient; this potential is monitored using the externally added fluorescent dye Safranine O. In the same system, transmembrane calcium fluxes are then detected using the (internally trapped) fluorescent dye Quin-2. The calcium-transport behavior of the channel-forming peptide alamethicin is shown to be potential dependent in this system, in contrast to calcium transport by the ionophore Br-A23187, which is unaffected by the potential. The observation of this potential-dependent behavior for alamethicin suggests that this vesicle system may be suitable for direct spectroscopic observation of the voltage-gating process.

Anionic phospholipids modulate peptide insertion into membranes.

While the insertion of a hydrophobic peptide or membrane protein segment into the bilayer can be spontaneous and driven mainly by the hydrophobic effect, anionic lipids, which comprise ca. 20% of biological membranes, provide a source of electrostatic attractions for binding of proteins/peptides into membranes. To unravel the interplay of hydrophobicity and electrostatics in the binding of peptides into membranes, we designed peptides de novo which possess the typical sequence Lys-Lys-Ala-Ala-Ala-X-Ala-Ala-Ala-Ala-Ala-X-Ala-Ala-Trp-Ala-Ala-X-Ala-Al a-Ala-Lys-Lys-Lys-Lys-amide, where X residues correspond to "guest" residues which encompass a range of hydrophobicity (Leu, Ile, Gly, and Ser). Circular dichroism spectra demonstrated that peptides were partially (40-90%) random in aqueous buffer but were promoted to form 100% alpha-helical structures by anionic lipid micelles. In neutral lipid micelles, only the relatively hydrophobic peptides (X = L and I) spontaneously adopted the alpha-helical conformation, but when 25% of negatively charged lipids were mixed in to mimic the content of anionic lipids in biomembranes, the less hydrophobic (X = S and G) peptides then formed alpha-helical conformations. Consistent with these findings, fluorescence quenching by the aqueous-phase quencher iodide indicated that in anionic (dimyristoylphosphatidylglycerol) vesicles, the peptide Trp residue was buried in the lipid vesicle hydrophobic core, while in neutral (dimyristoylphosphatidylcholine) vesicles, only hydrophobic (X = L and I) peptides were shielded from the aqueous solution. Trp emission spectra of peptides in the presence of phospholipids doxyl-labeled at the 5-, 7-, 10-, 12-, and 16-fatty acid positions implied not only a transbilayer orientation for inserted peptides but also that mixed peptide populations (transbilayer + surface-associated) may arise. Overall results suggest that for hydrophobic peptides with segmental threshold hydrophobicity below that which promotes spontaneous membrane insertion, primary electrostatic attractions provided by anionic phospholipids become essential for peptide binding and insertion to membranes.

Evidence for a folded conformation of methionine- and leucine-enkephalin in a membrane environment.

Transfer of an aqueous-soluble peptide hormone or neurotransmitter such as [Met]- or [Leu]enkephalin (Tyr1-Gly2-Gly3-Phe4-Met5(Leu5)), to the lipid-rich environment of its membrane-embedded receptor protein may convert the peptide into a ("bioactive") conformation required for eliciting biological activity. We have examined by high-resolution nuclear magnetic resonance (NMR) spectroscopy the conformational parameters of free enkephalin in aqueous solution versus those of enkephalin bound to lysophosphatidylcholine micelles using two approaches: 1) exchange rates, line broadening, coupling constants, and chemical shift changes of enkephalin backbone peptide N-H protons were measured for free and membrane-bound peptide in H2O (360 MHz, pH 5.6, 20 degrees C). A selective upfield shift observed for the Met5(Leu5) N-H proton upon lipid binding was interpreted in terms of its incorporation into an intramolecular H-bond. 2) 13C chemical shift changes induced by the shift reagent praseodymium nitrate (Pr(NO3)3) were compared in the presence and absence of lipid micelles. Significant changes occurring in Gly2 carbon atoms in membrane-bound enkephalin suggested the relative proximity of this residue to the Pr3+ atom (bound to the Met5(Leu5) COOH-terminal carboxylate 4 residues away). These combined results, in conjunction with studies on the specific interactions of enkephalin substituents with the micelles (Deber, C. M., and Behnam, B. A., (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 61-65) suggest that enkephalin folds into an intramolecularly H-bonded beta-turn structure (with an H-bond between Gly2 C = O and Met5 NH) in the lipid environment. Such folding could facilitate the positioning of strategic residues in vivo as the hormone diffuses toward its receptor.

Structure and function of the proline-rich region of myelin basic protein.

Myelin basic protein (MBP)--the major extrinsic membrane protein of central nervous system myelin--from several species contains a rarely encountered highly conserved triproline segment as residues 99-101 of its 170-residue sequence. Cis peptide bonds are known to arise at X-Pro junctions in proteins and may be of functional significance in protein folding, chain reversal, and/or maintenance of tertiary structure. We have examined the conformation of this proline-rich region using principally 13C nuclear magnetic resonance spectroscopy (125 MHz) both in intact bovine MBP and in several MBP fragment peptides which we synthesized, including octapeptide 97-104 (Arg-Thr-Pro-Pro-Pro-Ser-Gln-Gly). Results suggested an all-trans conformation in aqueous solution for the triproline segment in MBP hexapeptide (99-104), heptapeptide (98-104), and octapeptide. Comparison with the 13C spectrum of intact MBP (125 MHz) suggested that the proline-rich region, as well as all other X-Pro MBP peptide junctures, was also essentially all trans in aqueous solution. Although experiments in which octapeptide 97-104 was bound to a lipid preparation (4:1 dipalmitoylphosphatidylcholine/dimyristoylphosphatidic acid) demonstrated that cis-proline bonds do arise (to the extent of ca. 5%) in the membrane environment, a role of linear chain propagation is suggested for the triproline segment of myelin basic protein.