NMR structures and localization of the potential fusion peptides and the pre-transmembrane region of SARS-CoV: Implications in membrane fusion

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) poses a serious public health hazard. The S2 subunit of the S glycoprotein of SARS-CoV carries out fusion between the virus and the host cells. However, the exact mechanism of the cell fusion process is not well understood. Current model suggests that a conformational transition, upon receptor recognition, of the two heptad core regions of S2 may expose the hydrophobic fusogenic peptide or fusion peptide for membrane insertion. Three regions of the S2 subunit have been proposed to be involved in cell–cell fusion. The N-terminal fusion peptide (FP, residues 770–788), an internal fusion peptide (IFP, residues 873–888) and the pre-transmembrane region (PTM, residues 1185–1202) demonstrated interactions with model lipid membranes and potentially involved in the fusion process. Here, we have determined atomic resolution structures of these three peptides in DPC detergent micelles by solution NMR. FP assumes α-helical conformation with significant distortion at the central Gly residues; enabling a close packing among sidechains of aromatic residues including W, Y and F. The 3-D structure of PMT is characterized by a helix–loop–helix with extensive aromatic interactions within the helices. IFP adopts a rather straight α-helical conformation defined by packing among sidechains of aromatic and aliphatic residues. Paramagnetic spin labeled NMR has demonstrated surface localization of PMT whereas FP and IFP inserted into the micelles. Collectively, data presented in this study will aid in understanding fusion mechanism of SARS-CoV.     

Interaction of Influenza Virus Fusion Peptide with Lipid Membranes: Effect of Lysolipid

The effect of lysophosphatidylcholine (LPC) on lipid vesicle fusion and leakage induced by influenza virus fusion peptides and the peptide interaction with lipid membranes were studied by using fluorescence spectroscopy and monolayer surface tension measurements. It was confirmed that the wild-type fusion peptide-induced vesicle fusion rate increased several-fold between pH 7 and 5, unlike a mutated peptide, in which valine residues were substituted for glutamic acid residues at positions 11 and 15. This mutated peptide exhibited a much greater ability to induce lipid vesicle fusion and leakage but in a less pH-dependent manner compared to the wild-type fusion peptide. The peptide-induced vesicle fusion and leakage were well correlated with the degree of interaction of these peptides with lipid membranes, as deduced from the rotational correlation time obtained for the peptide tryptophan fluorescence. Both vesicle fusion and leakage induced by the peptides were suppressed by LPC incorporated into lipid vesicle membranes in a concentration-dependent manner. The rotational correlation time associated with the peptide’s tryptophan residue, which interacts with lipid membranes containing up to 25 mole % LPC, was virtually the same compared to lipid membranes without LPC, indicating that LPC-incorporated membrane did not affect the peptide interaction with the membrane. The adsorption of peptide onto a lipid monolayer also showed that the presence of LPC did not affect peptide adsorption.     

The polar region consecutive to the HIV fusion peptide participates in membrane fusion

The fusion peptide of HIV-1 gp41 is formed by the 16 N-terminal residues of the protein. This 16-amino acid peptide, in common with several other viral fusion peptides, caused a reduction in the bilayer to hexagonal phase transition temperature of dipalmitoleoylphosphatidylethanolamine (T(H)), suggesting its ability to promote negative curvature in membranes. Surprisingly, an elongated peptide corresponding to the 33 N-terminal amino acids raised T(H), although it was more potent than the 16-amino acid fusion peptide in inducing lipid mixing with large unilamellar liposomes of 1:1:1 dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine/choleste rol. The 17-amino acid C-terminal fragment of the peptide can induce membrane fusion by itself, if it is anchored to a membrane by palmitoylation of the amino terminus, indicating that the additional 17 hydrophilic amino acids contribute to the fusogenic potency of the peptide. This is not solely a consequence of the palmitoylation, as a random peptide with the same amino acid composition with a palmitoyl anchor was less potent in promoting membrane fusion and palmitic acid itself had no fusogenic activity. The 16-amino acid N-terminal fusion peptide and the longer 33-amino acid peptide were labeled with NBD. Fluorescence binding studies indicate that both peptides bind to the membrane with similar affinities, indicating that the increased fusogenic activity of the longer peptide was not a consequence of a greater extent of membrane partitioning. We also determined the secondary structure of the peptides using FTIR spectroscopy. We find that the amino-terminal fusion peptide is inserted into the membrane as a beta-sheet and the 17 C-terminal amino acids lie on the surface of the membrane, adopting an alpha-helical conformation. It was further demonstrated with the use of rhodamine-labeled peptides that the 33-amino acid peptide self-associated in the membrane while the 16-amino acid N-terminal peptide did not. Thus, the 16-amino acid N-terminal fusion peptide of HIV inserts into the membrane and, like other viral fusion peptides, lowers T(H). In addition, the 17 consecutive amino acids enhance the fusogenic activity of the fusion peptide presumably by promoting its self-association.     

The Structural Dynamics of the Flavivirus Fusion Peptide–Membrane Interaction

Membrane fusion is a crucial step in flavivirus infections and a potential target for antiviral strategies. Lipids and proteins play cooperative roles in the fusion process, which is triggered by the acidic pH inside the endosome. This acidic environment induces many changes in glycoprotein conformation and allows the action of a highly conserved hydrophobic sequence, the fusion peptide (FP). Despite the large volume of information available on the virus-triggered fusion process, little is known regarding the mechanisms behind flavivirus–cell membrane fusion. Here, we evaluated the contribution of a natural single amino acid difference on two flavivirus FPs, FLA_G (⁹⁸DRGWGNGCGLFGK¹¹⁰) and FLA_H (⁹⁸DRGWGNHCGLFGK¹¹⁰), and investigated the role of the charge of the target membrane on the fusion process. We used an in silico approach to simulate the interaction of the FPs with a lipid bilayer in a complementary way and used spectroscopic approaches to collect conformation information. We found that both peptides interact with neutral and anionic micelles, and molecular dynamics (MD) simulations showed the interaction of the FPs with the lipid bilayer. The participation of the indole ring of Trp appeared to be important for the anchoring of both peptides in the membrane model, as indicated by MD simulations and spectroscopic analyses. Mild differences between FLA_G and FLA_H were observed according to the pH and the charge of the target membrane model. The MD simulations of the membrane showed that both peptides adopted a bend structure, and an interaction between the aromatic residues was strongly suggested, which was also observed by circular dichroism in the presence of micelles. As the FPs of viral fusion proteins play a key role in the mechanism of viral fusion, understanding the interactions between peptides and membranes is crucial for medical science and biology and may contribute to the design of new antiviral drugs.     

Conformation of a synthetic antigenic peptide from HIV-1 p24 protein induced by ionic micelles

We studied the interaction of the peptide AAMQMLKETINEEAAEWDRVHPVHAGPIA from the HIV-1 p24 protein in the presence of SDS (anionic) and CTABr (cationic) micelles at pH 7.0 by circular dichroism, fluorescence, and electron spin resonance (ESR). The micelles induced secondary structure as well as a blue shift in the tryptophan fluorescence emission, indicating an interaction between the peptide and the micelles. However, different contents of secondary structure elements were found when the peptide interacts with SDS or CTABr micelles. Steady-state anisotropy indicates a constraint on the rotational mobility of the tryptophan residue of the peptide upon interaction with micelles. ESR studies pointed to different locations for the peptide in either micelle. Our results suggested that at least part of the peptide might be located at the hydrophobic core of the CTABr micelles, probably at the C-terminal region, while it is more inserted into the SDS micelles.     

The membrane-proximal tryptophan-rich region of the HIV glycoprotein, gp41, forms a well-defined helix in dodecylphosphocholine micelles

The membrane-proximal tryptophan-rich region of the HIV transmembrane glycoprotein, gp41, plays an important role in the membrane fusion reaction. Using NMR spectroscopy, we have studied the tertiary structure of a synthetic 19-residue amidated peptide (NH2-KWASLWNWFNITNWLWYIK-CONH2) corresponding to this region in membrane-mimetic environments. Initial experiments in sodium dodecyl sulfate/H2O micelles and trifluoroethanol gave poor results, because of low solubility. However, in dodecylphosphocholine micelles, we obtained excellent 500 and 800 MHz NMR spectra, suggesting that the peptide has a preference for a zwitterionic membrane-like environment. The final NMR structures demonstrated a well-defined helical peptide with a backbone rmsd of 0.47 +/- 0.18 A. Four of the five tryptophan residues, as well as the tyrosine residue, formed a "collar" of aromatic residues along the axial length of the helix. By analogy to related tryptophan-rich antimicrobial peptides, the structure indicates that the aromatic residues of the HIV peptide are positioned within the membrane-water interface of a phospholipid bilayer. This is confirmed by the observation of direct NOEs between the aromatic residues of the peptide to the headgroup and interfacial protons of prototonated dodecylphosphocholine. The bulk of the polar residues are positioned on one face of this structure, with the hydrophobic phenylalanine side chain on the opposing face, forming an amphipathic structure. This work shows that the Trp-rich membrane-proximal region of HIV and related viruses can bind to the surfaces of zwitterioninc membranes in a "Velcro-like" manner.     

Structure of the antimicrobial peptide tritrpticin bound to micelles: a distinct membrane-bound peptide fold

Tritrpticin is a member of the cathelicidin family, a group of diverse antimicrobial peptides found in neutrophil granules. The three Trp and four Arg residues in the sequence VRRFPWWWPFLRR make this a Trp-rich cationic peptide. The structure of tritrpticin bound to membrane-mimetic sodium dodecyl sulfate micelles has been determined using conventional two-dimensional NMR methods. It forms two adjacent turns around the two Pro residues, a distinct fold for peptide-membrane interaction. The first turn involves residues 4-7, followed immediately by a second well-defined 3(10)-helical turn involving residues 8-11. The hydrophobic residues are clustered together and are clearly separated from the basic Arg residues, resulting in an amphipathic structure. Favorable interactions between the unusual amphipathic fold and the micelle surface are probably key to determining the peptide structure. NMR studies of the peptide in the micelle in the presence of the spin-label 5-doxylstearic acid determined that tritrpticin lies near the surface of the micelle, where its many aromatic side chains appear to be equally partitioned into the hydrophilic-hydrophobic interface. Additional fluorescence studies confirmed that the tryptophan residues are inserted into the micelle and are partially protected from the effects of the soluble fluorescence quencher acrylamide.     

Interaction with membrane mimics of transmembrane fragments 16 and 17 from the human multidrug resistance ABC transporter 1 (hMRP1/ABCC1) and two of their tryptophan variants

The human multidrug resistance-associated protein 1 (hMRP1/ABCC1) belongs to the ATP-binding cassette transporter superfamily. Together with P-glycoprotein (ABCB1) and the breast cancer resistance protein (BCRP/ABCG2), hMRP1 confers resistance to a large number of structurally diverse drugs. The current topological model of hMRP1 includes two cytosolic nucleotide-binding domains and 17 putative transmembrane (TM) helices forming three membrane-spanning domains. Mutagenesis and labeling studies have shown TM16 and TM17 to be important for function. We characterized the insertion of the TM16 fragment into dodecylphosphocholine (DPC) or n-dodecyl-β-d-maltoside (DM) micelles as membrane mimics and extended our previous work on TM17 (Vincent et al., 2007, Biochim. Biophys. Acta 1768, 538). We synthesized TM16 and TM17, with the Trp residues, W1198 in TM16 and W1246 in TM17, acting as an intrinsic fluorescent probe, and TM16 and TM17 Trp variants, to probe different positions in the peptide sequence. We assessed the interaction of peptides with membrane mimics by evaluating the increase in fluorescence intensity resulting from such interactions. In all micelle-bound peptides, the tryptophan residue appeared to be located, on average, in the head group micelle region, as shown by its fluorescence spectrum. Each tryptophan residue was partially accessible to both acrylamide and the brominated acyl chains of two DM analogs, as shown by fluorescence quenching. Tryptophan fluorescence lifetimes were found to depend on the position of the tryptophan residue in the various peptides, probably reflecting differences in local structures. Far UV CD spectra showed that TM16 contained significant β-strand structures. Together with the high Trp correlation times, the presence of these structures suggests that TM16 self-association may occur at the interface. In conclusion, this experimental study suggests an interfacial location for both TM16 and TM17 in membrane mimics. In terms of overall hMRP1 structure, the experimentally demonstrated amphipathic properties of these TM are consistent with a role in the lining of an at least partly hydrophilic transport pore, as suggested by the currently accepted structural model, the final structure being modified by interaction with other TM helices.     

A minor beta-structured conformation is the active state of a fusion peptide of vesicular stomatitis virus glycoprotein.

Entry of enveloped animal viruses into their host cells always depends on a step of membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. In a previous work, we identified a specific sequence in the VSV G protein, comprising the residues 145-164, directly involved in membrane interaction and fusion. In the present work we studied the interaction of pep[145-164] with membranes using NMR to solve the structure of the peptide in two membrane-mimetic systems: SDS micelles and liposomes composed of phosphatidylcholine and phosphatidylserine (PC:PS vesicles). The presence of medium-range NOEs showed that the peptide has a tendency to form N- and C-terminal helical segments in the presence of SDS micelles. Analysis of the chemical shift index indicated helix-coil equilibrium for the C-terminal helix under all conditions studied. At pH 7.0, the N-terminal helix also displayed a helix-coil equilibrium when pep[145-164] was free in solution or in the presence of PC:PS. Remarkably, at the fusogenic pH, the region of the N-terminal helix in the presence of SDS or PC:PS presented a third conformational species that was in equilibrium with the helix and random coil. The N-terminal helix content decreases pH and the minor beta-structured conformation becomes more prevalent at the fusogenic pH. These data point to a beta-conformation as the fusogenic active structure-which is in agreement with the X-ray structure, which shows a beta-hairpin for the region corresponding to pep[145-164].     

Structure of the bovine antimicrobial peptide indolicidin bound to dodecylphosphocholine and sodium dodecyl sulfate micelles

Indolicidin is a cationic, 13-residue antimicrobial peptide (ILPWKWPWWPWRR-NH(2)) which is unusually rich in tryptophan and proline. Its antimicrobial action involves the bacterial cytoplasmic membrane. Fluorescence and circular dichroism spectra demonstrated the structural similarity of indolicidin in complexes with large unilamellar phospolipid vesicles and with detergent micelles. The structure of indolicidin bound to zwitterionic dodecylphosphocholine (DPC) and anionic sodium dodecyl sulfate (SDS) micelles was determined using NMR methods and shown to represent a unique membrane-associated peptide structure. The backbone structure in DPC, well defined between residues 3 and 11, was extended, with two half-turns at residues Lys-5 and Trp-8. The backbone structure in SDS, well defined between residues 5 and 11, was also extended, but lacked the bend in the C-terminal half. Indolicidin in complexes with DPC had a central hydrophobic core composed of proline and tryptophan, which was bracketed by positively charged regions near the peptide termini. The tryptophan side chains, with one exception, folded flat against the peptide backbone, thus giving the molecule a wedge shape. Indolicidin in complexes with SDS had an arrangement of hydrophobic and cationic regions similar to that found in the presence of DPC. The tryptophan side chains were less well defined than for indolicidin in DPC and extended away from the peptide backbone. The preferred location of indolicidin in DPC micelles and lipid bilayers, analyzed using spin-label probes, was at the membrane interface.     

NMR structure and localization of a large fragment of the SARS-CoV fusion protein: Implications in viral cell fusion

The lethal Coronaviruses (CoVs), Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV) and most recently Middle East Respiratory Syndrome Coronavirus, (MERS-CoV) are serious human health hazard. A successful viral infection requires fusion between virus and host cells carried out by the surface spike glycoprotein or S protein of CoV. Current models propose that the S2 subunit of S protein assembled into a hexameric helical bundle exposing hydrophobic fusogenic peptides or fusion peptides (FPs) for membrane insertion. The N-terminus of S2 subunit of SARS-CoV reported to be active in cell fusion whereby FPs have been identified. Atomic-resolution structure of FPs derived either in model membranes or in membrane mimic environment would glean insights toward viral cell fusion mechanism. Here, we have solved 3D structure, dynamics and micelle localization of a 64-residue long fusion peptide or LFP in DPC detergent micelles by NMR methods. Micelle bound structure of LFP is elucidated by the presence of discretely folded helical and intervening loops. The C-terminus region, residues F42-Y62, displays a long hydrophobic helix, whereas the N-terminus is defined by a short amphipathic helix, residues R4-Q12. The intervening residues of LFP assume stretches of loops and helical turns. The N-terminal helix is sustained by close aromatic and aliphatic sidechain packing interactions at the non-polar face. ¹⁵N{¹H}NOE studies indicated dynamical motion, at ps-ns timescale, of the helices of LFP in DPC micelles. PRE NMR showed that insertion of several regions of LFP into DPC micelle core. Together, the current study provides insights toward fusion mechanism of SARS-CoV.     

Membrane interactions of cell-penetrating peptides probed by tryptophan fluorescence and dichroism techniques: correlations of structure to cellular uptake

This work reports on the binding and conformation of a series of CPPs in the bilayer membranes of large unilamellar vesicles and the effect of the presence of cholesterol. We show a negative correlation between alpha-helical structure and uptake efficiency for penetratin peptides where the two central arginine residues of penetratin are thought to be important for breaking the secondary structure. Penetratin alpha-helicity is also reduced upon incorporation of cholesterol into the membrane. Flow linear dichroism in the far-UV region shows that the penetratin peptides adopt a preferential orientation of the alpha-helix parallel to the bilayer, and the linear dichroism (LD) spectrum in the aromatic region indicates that the tryptophan residues are preferentially oriented parallel to the membrane. The Tat analogue TatP59W and the oligoarginine R7W, which are more efficient CPPs than penetratin, bind to membranes as random coils and do not show any orientation in LD, again indicating that alpha-helicity reduces uptake efficiency. Further, we observe large variations in tryptophan quantum yields for the five CPPs in this study and discuss this in terms of the ability to cause lipid rearrangement. Binding isotherms show that cholesterol increases the affinity of the peptide for the membrane, but tryptophan fluorescence lifetimes are essentially unaltered by incorporation of as much as 40 mol % cholesterol into the membrane, suggesting the absence of specific peptide-cholesterol interactions. Fluorescence emission maxima are insensitive to cholesterol and indicate that the peptide is positioned in the headgroup region. The results on peptide-membrane interactions are discussed in terms of possible uptake mechanisms.     

The study of the conformation and interaction of two tachykinin peptides in membrane mimicking systems by NMR spectroscopy and pulsed field gradient diffusion

Pulsed‐field gradient diffusion has been used to study the binding of two tachykinin peptides, [Tyr⁸]‐substance P (SP) and [Tyr⁰]‐neurokinin A (NKA) to two membrane‐mimicking micelles, dodecylphosphocholine, and sodium dodecylsulfate. The structure of these peptides bound to the micelles have also been studied by using two‐dimensional nmr and restrained simulated annealing calculations. No major difference in the structures of each peptide in the two micellar media was found. The difference between the micelle‐bound structure of [Tyr⁸]SP and that of SP was also minor. The longer helical conformation on the C‐terminus for [Tyr⁰]NKA was observed, compared with that for NKA. The relationship between the difference in the biological potencies of [Tyr⁸]SP and SP and the differences in their structure, especially the interaction of the side chains of the two aromatic residues, and the difference in their binding affinities to membrane was discussed. In addition, differences between the result of restrained molecular dynamics simulations of [Tyr⁸]SP in the presence of an explicit micelle and the present results were observed and discussed. © 1999 John Wiley & Sons, Inc. Biopoly 50: 555–568, 1999     

Interaction of adrenocorticotropin peptides with microheterogeneous systems — A fluorescence study

Adrenocorticotropin (ACTH) and α-melanocyte stimulating hormone (α-MSH) are peptides which present many physiological effects related to pigmentation, motor and sexual behavior, learning and memory, analgesia, anti-inflammatory and antipyretic processes. The 13 amino acid residues of α-MSH are the same initial sequence of ACTH and due to the presence of a tryptophan residue in position 9 of the peptide chain, fluorescence techniques could be used to investigate the conformational properties of the hormones in different environments and the mechanisms of interaction with biomimetic systems like sodium dodecyl sulphate (SDS) micelles, sodium dodecyl sulphate-poly(ethylene oxide) (SDS-PEO) aggregates and neutral polymeric micelles. In buffer solution, fluorescence parameters were typical of peptides containing tryptophan exposed to the aqueous medium and upon addition of surfactant and polymer molecules, the gradual change of those parameters demonstrated the interaction of the peptides with the microheterogeneous systems. From time-resolved experiments it was shown that the interaction proceeded with conformational changes in both peptides, and further information was obtained from quenching of Trp fluorescence by a family of N-alkylpyridinium ions, which possess affinity to the microheterogeneous systems dependent on the length of the alkyl chain. The quenching of Trp fluorescence was enhanced in the presence of charged micelles, compared to the buffer solution and the accessibility of the fluorophore to the quencher was dependent on the peptide and the alkylpyridinium: in ACTH(1–21) highest collisional constants were obtained using ethylpyridinium as quencher, indicating a location of the residue in the surface of the micelle, while in α-MSH the best quencher was hexylpyridinium, indicating insertion of the residue into the non-polar region of the micelles. The results had shown that the interaction between the peptides and the biomimetic systems where driven by combined electrostatic and hydrophobic effects: in ACTH(1–24) the electrostatic interaction between highly positively charged C-terminal and negatively charged surface of micelles and aggregates predominates over hydrophobic interactions involving residues in the central region of the peptide; in α-MSH, which presents one residual positive charge, the hydrophobic interactions are relevant to position the Trp residue in the non-polar region of the microheterogeneous systems.     

Structural and thermodynamic analyses of the interaction between melittin and lipopolysaccharide

Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, is the very first site of interactions with the antimicrobial peptides. In this work, we have determined a solution conformation of melittin, a well-known membrane active amphiphilic peptide from honey bee venom, by transferred nuclear Overhauser effect (Tr-NOE) spectroscopy in its bound state with lipopolysaccharide. The LPS bound conformation of melittin is characterized by a helical structure restricted only to the C-terminus region (residues A15-R24) of the molecule. Saturation transfer difference (STD) NMR studies reveal that several C-terminal residues of melittin including Trp19 are in close proximity with LPS. Isothermal titration calorimetry (ITC) data demonstrates that melittin binding to LPS or lipid A is an endothermic process. The interaction between melittin and lipid A is further characterized by an equilibrium association constant (Ka) of 2.85 x 10(6) M(-1) and a stoichiometry of 0.80, melittin/lipid A. The estimated free energy of binding (delta G0), -8.8 kcal mol(-1), obtained from ITC experiments correlates well with a partial helical structure of melittin in complex with LPS. Moreover, a synthetic peptide fragment, residues L13-Q26 or mel-C, derived from the C-terminus of melittin has been found to contain comparable outer membrane permeabilizing activity against Escherichia coli cells. Intrinsic tryptophan fluorescence experiments of melittin and mel-C demonstrate very similar emission maxima and quenching in presence of LPS micelles. The Red Edge Excitation Shift (REES) studies of tryptophan residue indicate that both peptides are located in very similar environment in complex with LPS. Collectively, these results suggest that a helical conformation of melittin, at its C-terminus, could be an important element in recognition of LPS in the outer membrane.     

Peptide conformational changes induced by tryptophan-phosphocholine interactions in a micelle

Sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC) micelles are often used to mimic the membrane- or receptor-bound states of peptides in NMR studies. From the present examination of a 26-residue analog of exendin-4 (TrEX4) by NMR and CD in water, aqueous 30% trifluoroethanol (TFE), and bound to both SDS and DPC micelles, it is clear that these two lipid micelles can yield very different peptide structures. The Trp-cage fold (also observed in 30% TFE) is present when TrEX4 is bound to SDS micelles; however, tertiary structure is absent in the presence of DPC micelles. The loss of tertiary structure is attributed to an energetically favorable interaction (estimated as 2-3 kcal/mol) of the tryptophan side chain with the phosphocholine head groups. These dramatic structural differences suggest that care must be taken when using either SDS or DPC to mimic the membrane- or receptor-bound states.     

Secretin self-assembles and interacts spontaneously with phospholipids in vitro

Secretin, a 27-amino acid neuropeptide, is a member of the secretin/glucagon/vasoactive intestinal polypeptide (VIP) superfamily of amphipathic peptides. The peptide modulates gastrointestinal and neuronal function and is currently being evaluated for the treatment of autism. However, as most peptides, it has a short circulation half-life. Previously, we have shown that VIP self-assembles in aqueous environment and interacts with a biomimetic phospholipid membrane. These in vitro characteristics increase VIP half-life and bioactivity in vivo. The purpose of this study was to investigate whether secretin exhibits similar properties in vitro by forming micelles in aqueous solution and interacting with phospholipids. Results of this study demonstrated that secretin self-assembles to form micelles in HEPES buffer at 25 degrees C above approximately 0.4 microM. Additionally, secretin interacts with a biomimetic phospholipid membrane as indicated from a significant increase in membrane surface pressure (from 25.5 +/- 1.3 to 32.5 +/- 3.0, P < 0.05). Importantly, the peptide undergoes conformational transition from predominantly random coil in saline to alpha-helix in the presence of phospholipid, distearoyl-phosphatidylcholine-poly(ethylene) glycol (mol mass 2000) micelles. We suggest that these distinct biophysical attributes could modulate secretin bioactivity in vivo.     

NMR studies of aurein 1.2 analogs

Aurein 1.2 is an antimicrobial and anticancer peptide isolated from an Australian frog. To improve our understanding of the mechanism of action, two series of peptides were designed. The first series includes the N-terminal membrane anchor of bacterial glucose-specific enzyme IIA, aurein 1.2, and a newly identified aurein 1.2 analog from human LL-37 (LLAA). The order of antibacterial activity is LLAA>aurein 1.2>the membrane anchor (inactive). The structure of LLAA in detergent micelles was determined by (1)H NMR spectroscopy, including structural refinement by natural abundance (13)C(alpha), (13)C(beta), and (15)N chemical shifts. The hydrophobic surface area of the 3D structure is related to the retention time of the peptide on a reverse-phase HPLC column. The higher activity of LLAA compared to aurein 1.2 was attributed to additional cationic residues that enhance the membrane perturbation potential. The second peptide series was created by changing the C-terminal phenylalanine (F13) of aurein 1.2 to either phenylglycine or tryptophan. A closer or further location of the aromatic rings to the peptide backbone in the mutants relative to F13 is proposed to cause a drop in activity. Phenylglycine with unique chemical shifts may be a useful NMR probe for structure-activity relationship studies of antimicrobial peptides. To facilitate potential future use for NMR studies, random-coil chemical shifts for phenylglycine (X) were measured using the synthetic peptide GGXGG. Aromatic rings of phenylalanines in all the peptides penetrated 2-5 A below the lipid head group and are essential for membrane targeting as illustrated by intermolecular peptide-lipid NOE patterns.     

The aromatic domain of the coronavirus class I viral fusion protein induces membrane permeabilization: putative role during viral entry

Coronavirus (CoV) entry is mediated by the viral spike (S) glycoprotein, a class I viral fusion protein. During viral and target cell membrane fusion, the heptad repeat (HR) regions of the S2 subunit assume a trimer-of-hairpins structure, positioning the fusion peptide in close proximity to the C-terminal region of the ectodomain. The formation of this structure appears to drive apposition and subsequent fusion of viral and target cell membranes; however, the exact mechanism is unclear. Here, we characterize an aromatic amino acid rich region within the ectodomain of the S2 subunit that both partitions into lipid membranes and has the capacity to perturb lipid vesicle integrity. Circular dichroism analysis indicated that peptides analogous to the aromatic domains of the severe acute respiratory syndrome (SARS)-CoV, mouse hepatitis virus (MHV) and the human CoV OC43 S2 subunits, did not have a propensity for a defined secondary structure. These peptides strongly partitioned into lipid membranes and induced lipid vesicle permeabilization at peptide/lipid ratios of 1:100 in two independent leakage assays. Thus, partitioning of the peptides into the lipid interface is sufficient to disorganize membrane integrity. Our study of the S2 aromatic domain of three CoVs provides supportive evidence for a functional role of this region. We propose that, when aligned with the fusion peptide and transmembrane domains during membrane apposition, the aromatic domain of the CoV S protein functions to perturb the target cell membrane and provides a continuous track of hydrophobic surface, resulting in lipid-membrane fusion and subsequent viral nucleocapsid entry.     

Structural analysis and amphiphilic properties of a chemically synthesized mitochondrial signal peptide

Anionic phospholipids induce a marked conformational change in a synthetic peptide corresponding to residues 1-27 of pre-ornithine carbamyltransferase. The peptide designated, pO-(1-27)-peptide amide, becomes more alpha-helical in the presence of cardiolipin or dimyristoylphosphatidylglycerol but not in the presence of dimyristoylphosphatidylcholine. The greater helix-promoting action of anionic versus zwitterionic lipids is predicted by helix-coil transition theory. This statistical mechanical theory also predicts that a shorter peptide, N-acetyl-pO-(16-27)-peptide amide, has less helix-forming tendency, even in the presence of sodium dodecyl sulfate, despite the fact that it has a comparable number of positive charges. The N-acetyl-pO-(16-27)-peptide amide has no helical structure in buffer with or without dimyristoylphosphatidylglycerol but it has a small (5%) helical content in methanol. Thus, the ability of anionic lipids to promote helix formation requires more than the presence of cationic groups on the peptide. The angular dependence of the hydrophobic moment of the putative helical segment of pO-(1-27)-peptide amide demonstrates that any helical structure which is formed would have some amphiphilic character. The pO-(1-27)-peptide amide disrupts large lipid aggregates to form discoid micelles about 30 to 50 nm in diameter. The ability to lyse membranes into disc-shaped micelles is characteristic of peptides containing an amphiphathic helix. In the case of the mitochondrial signal peptide, this membrane-lytic behavior may contribute to the translocation of the protein into the organelle.