Structural properties of signal peptides and their membrane insertion

Structural properties of the amino acid sequences from 22 signal peptides have been analyzed and compared with peptides known to interact with biological membranes and liposomes, melittin, a lytic peptide of bee venom, and the non-polar C-terminal segment of cytochrome b5. All these peptides evidence a double amphipatic structure with an hydrophobic core of 9 to 24 amino acid residues and two charged polar ends. They all exhibit a high potential for making alpha-helix and, to a lesser degree, extended or beta-sheet conformation with low or negative potentials for making reverse turns or aperiodic conformation. A model of spontaneous insertion of these peptides into the lipid bilayer without specific surface receptor protein is proposed, where the two polar ends interact with each polar face of the lipid bilayer and the hydrophobic core inserts into the non-hydrogen bonding environment of the fatty acid side chains. This insertion could be the molecular trigger for ribophorin assembly around the signal peptide and subsequent attachment to the ribosome prior to the transfer of the polypeptide chain through the endoplasmic reticulum membrane.     

Perturbation of the hydrophobic core of lipid bilayers by the human antimicrobial peptide LL-37

LL-37 is a cationic, amphipathic alpha-helical antimicrobial peptide found in humans that kills cells by disrupting the cell membrane. To disrupt membranes, antimicrobial peptides such as LL-37 must alter the hydrophobic core of the bilayer. Differential scanning calorimetry and deuterium ((2)H) NMR experiments on acyl chain perdeuterated lipids demonstrate that LL-37 inserts into the hydrophobic region of the bilayer and alters the chain packing and cooperativity. The results show that hydrophobic interactions between LL-37 and the hydrophobic acyl chains are as important for the ability of this peptide to disrupt lipid bilayers as its electrostatic interactions with the polar headgroups. The (2)H NMR data are consistent with the previously determined surface orientation of LL-37 (Henzler Wildman, K. A., et al. (2003) Biochemistry 42, 6545) with an estimated 5-6 A depth of penetration of the hydrophobic face of the amphipathic helix into the hydrophobic interior of the bilayer. LL-37 also alters the material properties of lipid bilayers, including the area per lipid, hydrophobic thickness, and coefficient of thermal expansion in a manner that varies with lipid type and temperature. Comparison of the effect of LL-37 on 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC-d(31)) and 1,2-dimyristoyl-phosphatidylcholine (DMPC-d(54)) at different temperatures demonstrates the importance of bilayer order in determining the type and extent of disordering and disruption of the hydrophobic core by LL-37. One possible explanation, which accounts for both the (2)H NMR data presented here and the known surface orientation of LL-37 under identical conditions, is that bilayer order influences the depth of insertion of LL-37 into the hydrophobic/hydrophilic interface of the bilayer, altering the balance of electrostatic and hydrophobic interactions between the peptide and the lipids.     

Importance of hydrophobic matching for spontaneous insertion of a single-spanning membrane protein

In this study, we have investigated the effect of hydrophobic mismatch between the thickness of the membrane and a transmembrane segment of a protein that directly inserts into the membrane bilayer. For this purpose we used mutants of the single-spanning Pf3 coat protein that can spontaneously insert into Escherichia coli membrane vesicles and large unilamellar vesicles (LUVs). The thickness of the liposomal bilayer could be altered by using lipids with different acyl chain lengths or by incorporation of cholesterol. The insertion efficiency of the protein clearly depended on the bilayer thickness, with most efficient insertion under hydrophobic matching conditions. To discriminate between effects of length and hydrophobicity, mutants with different synthetic transmembrane segments were constructed. These mutants inserted into LUVs in a mismatch-dependent manner. However, in particular for longer and less hydrophobic mutants, most efficient insertion was generally observed in thinner bilayers than expected on the basis of hydrophobic matching.     

Biological activity and structural aspects of PGLa interaction with membrane mimetic systems

Peptidyl-glycine-leucine-carboxyamide (PGLa), isolated from granular skin glands of Xenopus laevis, is practically devoid of secondary structure in aqueous solution and in the presence of zwitterionic phospholipids, when added exogenously, but adopts an α-helix in the presence of anionic lipids. The peptide was shown to exhibit antifungal activity and to have antimicrobial activity towards both Gram-negative and Gram-positive bacteria. As a broad variety of peptides is found in the secretions of amphibian skin combinatorial treatment of PGLa and magainin 2 was studied showing enhanced activity by a heterodimer formation. Thus production of mutually recognizing peptides seems to be an effective way in nature to increase selective membrane activity. Biophysical studies on membrane mimics demonstrated that PGLa can discriminate between different lipid species, preferentially interacting with negatively charged lipids, which are major components of bacterial but not mammalian cell membranes. This emphasizes the role of electrostatic interactions as a major determinant to trigger the affinity of antimicrobial peptides towards bacterial membranes. PGLa induced the formation of a quasi-interdigitated phase in phosphatidylglycerol bilayers below their chain melting transition, which is due to the creation of voids below the peptide being aligned parallel to the membrane surface. In the fluid phase of phosphatidylglycerol the peptide inserts perpendicularly into the bilayer above a threshold concentration, which results in a hydrophobic mismatch of the peptide length and bilayer core for lipids ≤ C16. This mismatch is compensated by stretching of the acyl chains and in turn thickening of the bilayer demonstrating that membrane thinning cannot be taken generally as the hallmark of pore formation by antimicrobial peptides. Furthermore, PGLa was shown to affect membrane curvature strain of phosphatidylethanolamine, another main lipid component of bacterial membranes, where a cubic phase coexists with the fluid bilayer phase. Investigations on living Escherichia coli showed distinct changes in cell envelope morphology, when treated with the peptide. In a first stage loss of surface stiffness and consequently of topographic features was observed, followed in a second stage by permeabilization of the outer membrane and rupture of the inner (cytoplasmic) membrane supposedly by the mechanism(s) derived from model studies.     

Interaction between Alzheimer's Abeta(25-35) peptide and phospholipid bilayers: the role of cholesterol

There is mounting evidence that the lipid matrix of neuronal cell membranes plays an important role in the accumulation of beta-amyloid peptides into senile plaques, one of the hallmarks of Alzheimer's disease (AD). With the aim to clarify the molecular basis of the interaction between amyloid peptides and cellular membranes, we investigated the interaction between a cytotoxic fragment of Abeta(1-42), i.e., Abeta(25-35), and phospholipid bilayer membranes. These systems were studied by Electron Paramagnetic Resonance (EPR) spectroscopy, using phospholipids spin-labeled on the acyl chain. The effect of inclusion of charged phospholipids or/and cholesterol in the bilayer composition was considered in relation to the peptide/membrane interaction. The results show that Abeta(25-35) inserts in bilayers formed by the zwitterionic phospholipid dilauroyl phosphatidylcholine (DLPC), positioning between the outer part of the hydrophobic core and the external hydrophilic layer. This process is not significantly influenced by the inclusion of the anionic phospholipid phosphatidylglycerol (DLPG) in the bilayer, indicating the peptide insertion to be driven by hydrophobic rather than electrostatic interactions. Cholesterol plays a fundamental role in regulating the peptide/membrane association, inducing a membrane transition from a fluid-disordered to a fluid-ordered phase. At low cholesterol content, in the fluid-disordered phase, the insertion of the peptide in the membrane causes a displacement of cholesterol towards the more external part of the membrane. The crowding of cholesterol enhances its rigidifying effect on this region of the bilayer. Finally, the cholesterol-rich fluid-ordered membrane looses the ability to include Abeta(25-35).     

Cellular impermeability and uptake of biocides and antibiotics in gram-positive bacteria and mycobacteria

Gram-positive bacteria possess a permeable cell wall that usually does not restrict the penetration of antimicrobials. However, resistance due to restricted penetration can occur, as illustrated by vancomycin-intermediate resistant Staphylococcus aureus strains (VISA) which produce a markedly thickened cell wall. Alterations in these strains include increased amounts of nonamidated glutamine residues in the peptidoglycan and it is suggested that the resistance mechanism involves 'affinity trapping' of vancomycin in the thickened cell wall. VISA strains have reduced doubling times, lower sensitivity to lysostaphin and reduced autolytic activity, which may reflect changes in the D-alanyl ester content of the wall and membrane teichoic acids. Mycobacterial cell walls have a high lipid content, which is assumed to act as a major barrier to the penetration of antimicrobial agents. Relatively hydrophobic antibiotics such as rifampicin and fluoroquinolones may be able to cross the cell wall by diffusion through the hydrophobic bilayer composed of long chain length mycolic acids and glycolipids. Hydrophilic antibiotics and nutrients cannot diffuse across this layer and are thought to use porin channels which have been reported in many species of mycobacteria. The occurrence of porins in a lipid bilayer supports the view that the mycobacterial wall has an outer membrane analogous to that of gram-negative bacteria. However, mycobacterial porins are much less abundant than in the gram-negative outer membrane and allow only low rates of uptake for small hydrophilic nutrients and antibiotics.     

Solution structures and model membrane interactions of lactoferrampin, an antimicrobial peptide derived from bovine lactoferrin

Bovine lactoferrampin (LFampinB) has been identified as a novel antimicrobial peptide, which is derived from the N-terminal lobe of bovine lactoferrin. In this study, the solution structure of LFampinB bound to negatively charged sodium dodecyl sulphate micelles and zwitterionic dodecyl phosphocholine micelles was determined using 2-dimensional nuclear magnetic resonance (NMR) spectroscopy. The interaction between LFampinB and multilamellar phospholipid vesicles, containing choline and glycerol head groups, was examined using differential scanning calorimetry (DSC). In addition, the interaction between the N-terminal tryptophan residue and model membranes of varying composition was analyzed by fluorescence spectroscopy. LFampinB adopts an amphipathic alpha-helical conformation across the first 11 residues of the peptide but remains relatively unstructured at the C-terminus. The hydrophobic surface of the amphipathic helix is bordered by the side chains of Trp1 and Phe11, and is seen in both micelle-bound structures. The fluorescence results suggest that Trp1 inserts into the membrane at the lipid/water interface. The phenyl side chain of Phe11 is oriented in the same direction as the indole ring of Trp1, allowing these two residues to serve as anchors for the lipid bilayer. The DSC results also indicate that LFampinB interacts with glycerol head groups in multilamellar vesicles but has little effect on acyl chain packing. Our results support a two step model of antimicrobial activity where the initial attraction of LFampinB is mediated by the cluster of positive charges on the C-terminus followed by the formation of the N-terminal helix which binds to the surface of the bacterial lipid bilayer.     

Structure and dynamics of model pore insertion into a membrane

A cylindrical transmembrane molecule is constructed by linking hydrophobic sites selected from a coarse grain model. The resulting hollow tube assembly serves as a representation of a transmembrane channel, pore, or a carbon nanotube. The interactions of a coarse grain di-myristoyl-phosphatidyl-choline hydrated bilayer with both a purely hydrophobic tube and a tube with hydrophilic caps are studied. The hydrophobic tube rotates in the membrane and becomes blocked by lipid tails after a few tens of nanoseconds. The hydrophilic sites of the capped tube stabilize it by anchoring the tube in the lipid headgroup/water interfacial region of each membrane leaflet. The capped tube remains free of lipid tails. The capped tube spontaneously conducts coarse grain water sites; the free-energy profile of this process is calculated using three different methods and is compared to the barrier for water permeation through the lipid bilayer. Spontaneous tube insertion into an undisturbed lipid bilayer is also studied, which we reported briefly in a previous publication. The hydrophobic tube submerges into the membrane core in a carpetlike manner. The capped tube laterally fuses with the closest leaflet, and then, after plunging into the membrane interior, rotates to assume a transbilayer orientation. Two lipids become trapped at the end of the tube as it penetrates the membrane. The hydrophilic headgroups of these lipids associate with the lower tube cap and assist the tube in crossing the interior of the membrane. When the rotation is complete these lipids detach from the tube caps and fuse with the lower leaflet lipids.     

Solution structures and model membrane interactions of lactoferrampin, an antimicrobial peptide derived from bovine lactoferrin

Bovine lactoferrampin (LFampinB) has been identified as a novel antimicrobial peptide, which is derived from the N-terminal lobe of bovine lactoferrin. In this study, the solution structure of LFampinB bound to negatively charged sodium dodecyl sulphate micelles and zwitterionic dodecyl phosphocholine micelles was determined using 2-dimensional nuclear magnetic resonance (NMR) spectroscopy. The interaction between LFampinB and multilamellar phospholipid vesicles, containing choline and glycerol head groups, was examined using differential scanning calorimetry (DSC). In addition, the interaction between the N-terminal tryptophan residue and model membranes of varying composition was analyzed by fluorescence spectroscopy. LFampinB adopts an amphipathic alpha-helical conformation across the first 11 residues of the peptide but remains relatively unstructured at the C-terminus. The hydrophobic surface of the amphipathic helix is bordered by the side chains of Trp1 and Phe11, and is seen in both micelle-bound structures. The fluorescence results suggest that Trp1 inserts into the membrane at the lipid/water interface. The phenyl side chain of Phe11 is oriented in the same direction as the indole ring of Trp1, allowing these two residues to serve as anchors for the lipid bilayer. The DSC results also indicate that LFampinB interacts with glycerol head groups in multilamellar vesicles but has little effect on acyl chain packing. Our results support a two step model of antimicrobial activity where the initial attraction of LFampinB is mediated by the cluster of positive charges on the C-terminus followed by the formation of the N-terminal helix which binds to the surface of the bacterial lipid bilayer.     

Membrane structure and fusion-triggering conformational change of the fusion domain from influenza hemagglutinin

The N-terminal domain of the influenza hemagglutinin (HA) is the only portion of the molecule that inserts deeply into membranes of infected cells to mediate the viral and the host cell membrane fusion. This domain constitutes an autonomous folding unit in the membrane, causes hemolysis of red blood cells and catalyzes lipid exchange between juxtaposed membranes in a pH-dependent manner. Combining NMR structures determined at pHs 7.4 and 5 with EPR distance constraints, we have deduced the structures of the N-terminal domain of HA in the lipid bilayer. At both pHs, the domain is a kinked, predominantly helical amphipathic structure. At the fusogenic pH 5, however, the domain has a sharper bend, an additional 3(10)-helix and a twist, resulting in the repositioning of Glu 15 and Asp 19 relative to that at the nonfusogenic pH 7.4. Rotation of these charged residues out of the membrane plane creates a hydrophobic pocket that allows a deeper insertion of the fusion domain into the core of the lipid bilayer. Such an insertion mode could perturb lipid packing and facilitate lipid mixing between juxtaposed membranes.     

The lipopeptide antibiotic A21978C has a specific interaction with DMPC only in the presence of calcium ions

The A21978C group are lipopeptide antibiotics which kill Gram-positive bacteria only in the presence of calcium ions. The calcium requirement of the antibacterial activity of A21978C correlates well with an in vitro calcium-dependent insertion into phospholipid vesicles. In this paper the interaction of A21978C with phosphatidylcholine is investigated in mixed monomolecular films. The spontaneity of the antibiotic-lipid mixing was determined by calculating the free energy change. On a Ca2+ containing subphase there is a specific interaction between the components at all antibiotic-lipid ratios. This is not true on K+ subphases, where specific interactions never occur. On Mg2+ subphases specific interactions occur only in monolayers containing very little lipid. By analysing the fluorescence of the kynurenine residue we have followed the effects of two factors on the penetration of the antibiotic into lipid bilayer vesicles. Firstly, the phospholipid gel to liquid crystalline phase transition which in the absence of calcium leads to an exclusion of the antibiotic from the bilayer. This trend is completely reversed in the presence of Ca2+. Secondly, the role of this lipopeptide's lipid tail was clarified by use of a series of versions of increasing fatty acyl chain length. The results indicate that the interaction promoted by calcium is not simply a hydrophobic attraction between fatty acyl chains but is more likely to be a specific interaction between polar headgroups.     

Cytochrome c adsorption to supported, anionic lipid bilayers studied via atomic force microscopy

The adsorption of membrane-associated protein cytochrome c to anionic lipid bilayers of dioleoyl phosphatidylglycerol was studied in low ionic strength physiological buffer using atomic force microscopy. The bilayers were supported on polylysinated mica. The formation of stable, single lipid bilayers was confirmed by imaging and force spectroscopy. Upon addition of low concentrations of cytochrome c, protein molecules were not topographically visible on the lipid bilayer-buffer interface. However, the forces required to punch through the bilayer by indentation using the atomic force microscopy probe were significantly lower after protein adsorption, which suggest that the protein inserts into the bilayer. Moreover, the apparent thickness of the bilayer remained unchanged after cytochrome c adsorption. Yet, mass spectroscopy and visible light absorption spectroscopy confirmed the presence of cytochrome c in the lipid bilayers. These results suggest that 1), cytochrome c inserts into the bilayer and resides in its hydrophobic core; 2), cytochrome c insertion changes the mechanical properties of the bilayer significantly; and 3), bilayer force spectroscopy may be a useful tool in investigating lipid-protein interactions.     

Cooperation of transmembrane segments during the integration of a double-spanning protein into the ER membrane

While membrane insertion of single-spanning membrane proteins into the endoplasmic reticulum (ER) is relatively well understood, it is unclear how multi-spanning proteins integrate. We have investigated the cotranslational ER integration of a double-spanning protein that is derived from leader peptidase. Both transmembrane (TM) segments are inserted into the membrane by the Sec61 channel. While the first, long and hydrophobic TM segment (TM1) inserts into the lipid bilayer on its own, the second, shorter TM anchor (TM2) collaborates with TM1 during its integration. TM1 diffuses away from the Sec61 complex in the absence of TM2, but is close to Sec61 when TM2 arrives inside the channel. These data suggest that the exit of a weak TM segment from the Sec61 channel into the lipid phase can be facilitated by its interaction with a previously integrated strong and stabilizing TM anchor.     

The activation energy for insertion of transmembrane alpha-helices is dependent on membrane composition

The physical mechanisms that govern the folding and assembly of integral membrane proteins are poorly understood. It appears that certain properties of the lipid bilayer affect membrane protein folding in vitro, either by modulating helix insertion or packing. In order to begin to understand the origin of this effect, we investigate the effect of lipid forces on the insertion of a transmembrane alpha-helix using a water-soluble, alanine-based peptide, KKAAAIAAAAAIAAWAAIAAAKKKK-amide. This peptide binds to preformed 1,2-dioleoyl-l-alpha-phosphatidylcholine (DOPC) vesicles at neutral pH, but spontaneous transmembrane helix insertion directly from the aqueous phase only occurs at high pH when the Lys residues are de-protonated. These results suggest that the translocation of charge is a major determinant of the activation energy for insertion. Time-resolved measurements of the insertion process at high pH indicate biphasic kinetics with time constants of ca 30 and 430 seconds. The slower phase seems to correlate with formation of a predominantly transmembrane alpha-helical conformation, as determined from the transfer of the tryptophan residue to the hydrocarbon region of the membrane. Temperature-dependent measurements showed that insertion can proceed only above a certain threshold temperature and that the Arrhenius activation energy is of the order of 90 kJ mol(-1). The kinetics, threshold temperature and the activation energy change with the mole fraction of 1,2-dioleoyl-l-alpha-phosphatidylethanolamine (DOPE) introduced into the DOPC membrane. The activation energy increases with increasing DOPE content, which could reflect the fact that this lipid drives the bilayer towards a non-bilayer transition and increases the lateral pressure in the lipid chain region. This suggests that folding events involving the insertion of helical segments across the bilayer can be controlled by lipid forces.     

Interactions of cholesterol with lipid bilayers: the preferred configuration and fluctuations.

The free energy difference associated with the transfer of a single cholesterol molecule from the aqueous phase into a lipid bilayer depends on its final location, namely on its insertion depth and orientation within the bilayer. We calculated desolvation and lipid bilayer perturbation contributions to the water-to-membrane transfer free energy, thus allowing us to determine the most favorable location of cholesterol in the membrane and the extent of fluctuations around it. The electrostatic and nonpolar contributions to the solvation free energy were calculated using continuum solvent models. Lipid layer perturbations, resulting from both conformational restrictions of the lipid chains in the vicinity of the (rigid) cholesterol backbone and from cholesterol-induced elastic deformations, were calculated using a simple director model and elasticity theory, respectively. As expected from the amphipathic nature of cholesterol and in agreement with the available experimental data, our results show that at the energetically favorable state, cholesterol's hydrophobic core is buried within the hydrocarbon region of the bilayer. At this state, cholesterol spans approximately one leaflet of the membrane, with its OH group protruding into the polar (headgroup) region of the bilayer, thus avoiding an electrostatic desolvation penalty. We found that the transfer of cholesterol into a membrane is mainly driven by the favorable nonpolar contributions to the solvation free energy, whereas only a small opposing contribution is caused by conformational restrictions of the lipid chains. Our calculations also predict a strong tendency of the lipid layer to elastically respond to (thermally excited) vertical fluctuations of cholesterol so as to fully match the hydrophobic height of the solute. However, orientational fluctuations of cholesterol were found to be accompanied by both an elastic adjustment of the surrounding lipids and by a partial exposure of the hydrophobic cholesterol backbone to the polar (headgroup) environment. Our calculations of the molecular order parameter, which reflects the extent of orientational fluctuations of cholesterol in the membrane, are in good agreement with available experimental data.     

Folding and assembly of beta-barrel membrane proteins

Beta-barrel membrane proteins occur in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts. The membrane-spanning sequences of beta-barrel membrane proteins are less hydrophobic than those of alpha-helical membrane proteins, which is probably the main reason why completely different folding and membrane assembly pathways have evolved for these two classes of membrane proteins. Some beta-barrel membrane proteins can be spontaneously refolded into lipid bilayer model membranes in vitro. They may also have this ability in vivo although lipid and protein chaperones likely assist with their assembly in appropriate target membranes. This review summarizes recent work on the thermodynamic stability and the mechanism of membrane insertion of beta-barrel membrane proteins in lipid model and biological membranes. How lipid compositions affect folding and assembly of beta-barrel membrane proteins is also reviewed. The stability of these proteins in membranes is not as large as previously thought (<10 kcal/mol) and is modulated by elastic forces of the lipid bilayer. Detailed kinetic studies indicate that beta-barrel membrane proteins fold in distinct steps with several intermediates that can be characterized in vitro. Formation of the barrel is synchronized with membrane insertion and all beta-hairpins insert simultaneously in a concerted pathway.     

Breaching the Barrier: Quantifying Antibiotic Permeability across Gram-negative Bacterial Membranes

The double-membrane cell envelope of Gram-negative bacteria is a sophisticated barrier that facilitates the uptake of nutrients and protects the organism from toxic compounds. An antibiotic molecule must find its way through the negatively charged lipopolysaccharide layer on the outer surface, pass through either a porin or the hydrophobic layer of the outer membrane, then traverse the hydrophilic peptidoglycan layer only to find another hydrophobic lipid bilayer before it finally enters the cytoplasm, where it typically finds its target. This complex uptake pathway with very different physico-chemical properties is one reason that Gram-negative are intrinsically protected against multiple classes of antibiotic-like molecules, and is likely the main reason that in vitro target-based screening programs have failed to deliver novel antibiotics for these organisms. Due to the lack of general methods available for quantifying the flux of drugs into the cell, little is known about permeation rates, transport pathways and accumulation at the target sites for particular molecules. Here we summarize the current tools available for measuring antibiotic uptake across the different compartments of Gram-negative bacteria.     

Mechanism of alamethicin insertion into lipid bilayers.

Alamethicin adsorbs on the membrane surface at low peptide concentrations. However, above a critical peptide-to-lipid ratio (P/L), a fraction of the peptide molecules insert in the membrane. This critical ratio is lipid dependent. For diphytanoyl phosphatidylcholine it is about 1/40. At even higher concentrations P/L > or = 1/15, all of the alamethicin inserts into the membrane and forms well-defined pores as detected by neutron in-plane scattering. A previous x-ray diffraction measurement showed that alamethicin adsorbed on the surface has the effect of thinning the bilayer in proportion to the peptide concentration. A theoretical study showed that the energy cost of membrane thinning can indeed lead to peptide insertion. This paper extends the previous studies to the high-concentration region P/L > 1/40. X-ray diffraction shows that the bilayer thickness increases with the peptide concentration for P/L > 1/23 as the insertion approaches 100%. The thickness change with the percentage of insertion is consistent with the assumption that the hydrocarbon region of the bilayer matches the hydrophobic region of the inserted peptide. The elastic energy of a lipid bilayer including both adsorption and insertion of peptide is discussed. The Gibbs free energy is calculated as a function of P/L and the percentage of insertion phi in a simplified one-dimensional model. The model exhibits an insertion phase transition in qualitative agreement with the data. We conclude that the membrane deformation energy is the major driving force for the alamethicin insertion transition.     

A glycophospholipid covalently attached to the C-terminus of the Thy-1 glycoprotein

The Thy-1 glycoprotein is a very abundant cell surface molecule of rat thymocytes and neuronal cells with the properties of a molecule that inserts into the lipid bilayer. The hydrophobicity is due to a glycophospholipid component covalently attached to the carboxy group of the C-terminal cysteine residue. The mature glycoprotein does not contain a stretch of hydrophobic amino acids that could traverse the membrane bilayer. These findings present a new mode of membrane attachment for a cell surface molecule that can mediate lymphokine release and cell division after cross-linking by antibodies.     

Possible role of a PXXP central hinge in the antibacterial activity and membrane interaction of PMAP-23, a member of cathelicidin family

Cathelicidins are essential components of the innate immune system of mammals, providing them a weapon against microbial invasion. PMAP-23 adopting a helix-hinge-helix structure with a central PXXP motif is a member of the cathelicidin family and has potent killing activities against a broad spectrum of microbial organisms. Although the antimicrobial effect of PMAP-23 is believed to be mediated by membrane disruption, many details of this event remain unclear. Here, we try to characterize the interaction between PMAP-23 and membrane phospholipids, focusing on the function of the central PXXP motif. PMAP-PA, in which the Pro residues were substituted by Ala, had significantly more alpha-helical content than PMAP-23, but was less amphipathic and more damaging to human erythrocytes and zwitterionic liposomes. The observed differences in the structures and biological activities of PMAP-23 and PMAP-PA confirmed the functional importance of the central hinge PXXP motif, which enables PMAP-23 to adopt a well-defined amphipathic conformation along its entire length and to have selective antimicrobial activity. CD and Trp fluorescence studies using fragments corresponding to the two helical halves of PMAP-23 revealed that the N-terminal half binds to anionic phospholipids and is more stable than the C-terminal half. In addition, Trp fluorescence quench analyses revealed that the C-terminal helix inserts more deeply into the hydrophobic region of the membrane than the N-terminal helix. Finally, observations made using biosensor technology enabled us to distinguish between the membrane binding and insertion steps, substantiating a proposed kinetic mode of the peptide-membrane interaction in which PMAP-23 first attaches to the membrane via the N-terminal amphipathic helix, after which bending and/or swiveling of the PXXP motif enables insertion of the C-terminal helix into the lipid bilayer.