Conformations of yeast alpha-mating factor and analog peptides as bound to phospholipid bilayer. Correlation of membrane-bound conformation with physiological activity

The transferred nuclear Overhauser effects of yeast alpha-mating factor [(1-13)peptide] in the presence of various spin-labeled phosphatidylcholines in small unilamellar vesicles of perdeuterated phosphatidylcholine have been analyzed. From the analysis of the quenching effect by spin-labels, the depth of amino acid side chains of the mating factor in phospholipid bilayer has been elucidated. The Leu4 and Leu6 residues are buried deeply in the apolar region of the phospholipid bilayer while the hydrophilic residues such as Gln5 and Lys7 are in the shallow region of the bilayer. The interaction of the side chains of Trp1 and Trp3 residues of alpha-mating factor with the hydrophobic interior of the bilayer contributes to the binding of this peptide with the phosphatidylcholine bilayer. The conformation of des-Trp1-alpha-mating-factor [(2-13)peptide] in the membrane-bound state has been found to be similar to that of (1-13)peptide from the analysis of transferred nuclear Overhauser effects in the presence of mixed vesicles of perdeuterated phosphatidylcholine and perdeuterated phosphatidylserine. The incorporation of this acidic phospholipid in the vesicle remarkably enhances the binding of (1-13)peptide and analog peptides. However, such modifications that weaken the interaction with phospholipid bilayer (deletion of Trp1 and substitution of Trp3 by Gly or Ala) appreciably lower the physiological activity. Transferred nuclear Overhauser effect analyses have also been made of [DHis2]peptide, [DLeu6]peptide and [DLys7]peptide in the presence of the vesicles of perdeuterated phosphatidylcholine. The main-chain conformations of these three analogs in the membrane-bound state have been found to be similar to that of (1-13)peptide, although the side-chain conformations of the D-amino acid residues are naturally different from those of the L-amino acid ones. Thus, the physiological activities of the (1-13)peptide and a variety of analog peptides are found to correlate with the affinities to the phosphatidylcholine/phosphatidylserine membrane and with the molecular conformations in the membrane-bound state.     

N-terminal half of a mitochondrial presequence peptide takes a helical conformation when bound to dodecylphosphocholine micelles: a proton nuclear magnetic resonance study

Two-dimensional proton nuclear magnetic resonance (NMR) spectra of a synthetic peptide (p25) corresponding to the amino-terminus of the yeast mitochondrial cytochrome oxidase subunit IV precursor protein have been analyzed. Sequence-specific resonance assignments of the peptide have been made in the presence of micelles of a phospholipid analog, perdeuterated dodecylphosphocholine (DPC), with the aid of such techniques as HOHAHA, DQF-COSY, and NOESY. The interresidue nuclear Overhauser effects (NOEs) indicate that the N-terminal half of p25 (S3-F11) takes a helical structure while the C-terminal half does not take a regular secondary structure. Addition of DPC to the solution of p25 induced chemical shift changes only of the resonances from the residues in the N-terminal half, suggesting that the N-terminal half of p25 is directly involved in binding to DPC. The induced helical structure in the N-terminal half at a lipid-water interface may be important in the ability of this presequence to direct a "passenger" protein into mitochondria.     

Structural characterizations of fusion peptide analogs of influenza virus hemagglutinin. Implication of the necessity of a helix-hinge-helix motif in fusion activity

Infection by enveloped viruses initially involves membrane fusion between viral and host cell membranes. The fusion peptide plays a crucial role in triggering this reaction. To clarify how the fusion peptide exerts this specific function, we carried out biophysical studies of three fusion peptide analogs of influenza virus hemagglutinin HA2, namely E5, G13L, and L17A. E5 exhibits an activity similar to the native fusion peptide, whereas G13L and L17A, which are two point mutants of the E5 analog, possess much less fusion activity. Our CD data showed that the conformations of these three analogs in SDS micelles are pH-dependent, with higher alpha-helical contents at acidic pH. Tryptophan fluorescence emission experiments indicated that these three analogs insert deeper into lipid bilayers at acidic pH. The three-dimensional structure of the E5 analog in SDS micelles at pH 4.0 revealed that two segments, Leu(2)-Glu(11) and Trp(14)-Ile(18), form amphipathic helical conformations, with Gly(12)-Gly(13) forming a hinge. The hydrophobic residues in the N- and C-terminal helices form a hydrophobic cluster. At neutral pH, however, the C-terminal helix of Trp(14)-Ile(18) reduces dramatically, and the hydrophobic core observed at acidic pH is severely disrupted. We suggest that the disruption of the C-terminal helix renders the E5 analog fusion-inactive at neutral pH. Furthermore, the decrease of the hinge and the reduction of fusion activity in G13L reveal the importance of the hinge in fusion activity. Also, the decrease in the C-terminal helix and the reduction of fusion activity in L17A demonstrates the importance of the C-terminal helix in fusion activity. Based on these biophysical studies, we propose a model that illustrates the structural change of the HA2 fusion peptide analog and explains how the analog interacts with the lipid bilayer at different pH values.     

Novel lactoferrampin antimicrobial peptides derived from human lactoferrin

Human lactoferrampin is a novel antimicrobial peptide found in the cationic N-terminal lobe of the iron-binding human lactoferrin protein. The amino acid sequence that directly corresponds to the previously characterized bovine lactoferrin-derived lactoferrampin peptide is inactive on its own (WNLLRQAQEKFGKDKSP, residues 269-285). However, by increasing the net positive charge near the C-terminal end of human lactoferrampin, a significant increase in its antibacterial and Candidacidal activity was obtained. Conversely, the addition of an N-terminal helix cap (sequence DAI) did not have any appreciable effect on the antibacterial or antifungal activity of human lactoferrampin peptides, even though it markedly influenced that of bovine lactoferrampin. The solution structure of five human lactoferrampin variants was determined in SDS micelles and all of the structures display a well-defined amphipathic N-terminal helix and a flexible cationic C-terminus. Differential scanning calorimetry studies indicate that this peptide is capable of inserting into the hydrophobic core of a membrane, while fluorescence spectroscopy results suggest that a hydrophobic patch encompassing the single Trp and Phe residues as well as Leu, Ile and Ala side chains mediates the interaction between the peptide and the hydrophobic core of a phospholipid bilayer.     

High-resolution NMR studies of fibrinogen-like peptides in solution: structural basis for the bleeding disorder caused by a single mutation of Gly(12) to Val(12) in the A alpha chain of human fibrinogen Rouen

In human fibrinogen Rouen, which is the origin of a bleedin disorder, a single amino acid is mutated from Gly(12) to Val(12) in the A alpha chain. In the previous paper of this series, this mutation was predicted to disrupt the structure of fibrinogen-like peptides bound to bovine thrombin. The structural basis of this bleeding disorder has been further assessed by studying the interaction of the following Val(12)-substituted human fibrinogen-like peptides with bovine thrombin in aqueous solution by use of two-dimensional NMR spectroscopy (including TRNOE): Ala-Asp-Ser-Gly-Glu-Gly-Asp(7)-Phe-Leu-Ala- Glu-Val(12)-Gly-Gly-Val-Arg(16)-Gly(17)-Pro-Arg-Val-NH2 (F16), Ala-Asp-Ser-Gly-Glu-Gly-Asp(7)-Phe-Leu-Ala-Glu-Val(12)-Gly-Gly-Val- Arg(16) (tF16), Ala-Asp-Ser-Gly-Glu-Cys(Acm)-Asp(7)-Phe-Leu-Ala-Glu-Val(12)-Gly-Gly-Val- Arg(16)-Gly(17)-Pro-Arg-Val-Cys(Acm)-NH2 (F17), and Ala-Asp-Ser-Gly-Glu-Cys(Acm)-Asp(7)-Phe-Leu-Ala-Glu-Val(12)-Gly-Gly- Val-Arg(16) (tF17). Binding of thrombin to peptides F16 and F17, and hence to tF16 and tF17 as a result of the cleavage of the Arg(16)-Gly(17) peptide bond, broadens the proton resonances of residues Asp(7) to Arg(16), suggesting that thrombin interacts specifically with this sequence of residues. Medium- and long-range TRNOE's were observed between the NH proton of Asp(7) and the C beta H protons of Ala(10) and between the ring protons of Phe(8) and the C gamma H protons of Val(12) and Val(15) in complexes of thrombin with both tF16 and tF17. A strong TRNOE, in peptides tF16 and tF17, between the C beta H protons of Glu(11) and the backbone NH proton of Val(12) was also observed. However, TRNOE's between the ring protons of Phe(8) and the C alpha H protons of Gly(14) and between the C alpha H proton of Glu(11) and the NH proton of Gly(13), previously observed in the complex of thrombin with FpA, were absent in both peptides tF16 and tF17. From incorporation of TRNOE information into distance geometry calculations, Val(12) was found to disrupt the type II beta-turn involving Glu(11) and Gly(12) that is present in complexes of thrombin with normal fibrinogen-like peptides. The positions of Gly(13) and Gly(14) in the complex are also displaced, relative to the aromatic ring of Phe(8), by the Val(12) substitution. This altered geometry presumably affects the positioning of the Arg(16)-Gly(17) bond in the active site of thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Solution structure of the osteogenic 1-31 fragment of the human parathyroid hormone

The solution conformations of a selectively osteogenic 1-31 fragment of the human parathyroid hormone (hPTH), hPTH(1-31)NH(2), have been characterized by use of very high field NMR spectroscopy at 800 MHz. The combination of the CalphaH proton and (13)Calpha chemical shifts, (3)J(NH)(alpha) coupling constants, NH proton temperature coefficients, and backbone NOEs reveals that the hPTH(1-31)NH(2) peptide has well-formed helical structures localized in two distinct segments of the polypeptide backbone. There are also many characteristic NOEs defining specific side-chain/backbone and side-chain/side-chain contacts within both helical structures. The solution structure of hPTH(1-31)NH(2) contains a short N-terminal helical segment for residues 3-11, including the helix capping residues 3 and 11 and a long C-terminal helix for residues 16-30. The two helical structures are reinforced by well-defined capping motifs and side-chain packing interactions within and at both ends of these helices. On one face of the C-terminal helix, there are side-chain pairs of Glu22-Arg25, Glu22-Lys26, and Arg25-Gln29 that can form ion-pair and/or hydrogen bonding interactions. On the opposite face of this helix, there are characteristic hydrophobic interactions involving the aromatic side chain of Trp23 packing against the aliphatic side chains of Leu15, Leu24, Lys27, and Leu28. There is also a linear array of hydrophobic residues from Val2, to Leu7, to Leu11 and continuing on to residues His14 and Leu15 in the hinge region and to Trp23 in the C-terminal helix. Capping and hydrophobic interactions at the end of the N-terminal and at the beginning of the C-terminal helix appear to consolidate the helical structures into a V-shaped overall conformation for at least the folded population of the hPTH(1-31)NH(2) peptide. Stabilization of well-folded conformations in this linear 1-31 peptide fragment and possibly other analogues of human PTH may have a significant impact on the biological activities of the PTH peptides in general and specifically for the osteogenic/anabolic activities of bone-building PTH analogues.     

High-resolution NMR studies of fibrinogen-like peptides in solution: structure of a thrombin-bound peptide corresponding to residues 7-16 of the A alpha chain of human fibrinogen

The interaction of the following human fibrinogen-like peptides with bovine thrombin was studied by one- and two-dimensional NMR techniques in aqueous solution: acetyl-Phe(8)-Leu(9)-Ala(10)-Glu-(11)-Gly(12)-Gly(13)-Gly(14)-Val(15)-Ar g(16)- Gly(17)-Pro(18)-NHMe (F6), acetyl-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg(16) (tF6), acetyl-Asp(7)-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg(16)-Gly(17)-Pro- Arg(19)-Val(20)-NHMe (F8), and acetyl-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg(16) (tF8). At pH 5.3 and 25 degrees C, the Arg(16)-Gly(17) peptide bonds in both F6 and F8 were cleaved instantaneously in the presence of 0.5 mM thrombin, producing truncated peptides tF6 and tF8 and other peptide fragments. On the basis of observations of line broadening, thrombin was found to bind to the cleavage products, tF6 and tF8, of peptides F6 and F8. Peptide tF8 may have a higher affinity for thrombin than peptide tF6, as suggested by the more pronounced thrombin-induced line broadening on the proton resonances in peptide tF8. Transferred NOE (TRNOE) measurements were made of the complexes between thrombin and peptides tF6 and tF8. Medium- and long-range NOE interactions were found between the NH proton of Asp(7) and the C beta H protons of Ala(10), between the C alpha H proton of Glu(11) and the NH proton of Gly(13), and between the ring protons of Phe(8) and the C alpha H protons of Gly(14) and the C gamma H protons of Val(15). Sets of structures of the decapeptide tF8 were deduced by use of distance geometry calculations based on sequential and medium- and long-range TRNOEs from the thrombin-bound peptide. A predominant feature of these structures is the nonpolar cluster formed by the side chains of residues Phe(8), Leu(9), and Val(15) that are directly involved in binding to thrombin. This structural feature is brought about by an alpha-helical segment involving residues Phe(8)-Ala(10), followed by a multiple-turn structure involving residues Glu(11)-Val(15). These results provide an explanation for the observations that Asp(7), Phe(8), and Gly(12) are strongly conserved in mammalian fibrinogens and that the mutations of Asp(7) to Asn(7) and of Gly(12) to Val(12), result in delayed release of fibrinopeptide A, producing human bleeding disorders.     

ACTH binding to phospholipid bilayers: A 1H-NMR study of the 5-14, 1-14, and 1-24 derivatives

The interaction of the 5-14, 1-14, and 1-24 fragments of ACTH with sonicated phospholipid bilayers containing egg yolk phosphatidylcholine (EPC) either pure or mixed with 10 mole % phosphatidic acid (EPA), was investigated by proton nuclear magnetic resonance (¹H-nmr). The effects observed with zwitterionic EPC vesicles were small, indicating a low binding of the ACTH derivatives. The N-terminal aromatic resonances of the ACTH peptides were markedly broadened in the presence of negatively charged vesicles (EPC/EPA 9:1 M/M), while those of the C-terminal end were barely affected, showing that ACTH interacts with its N-terminal fragment. The choline resonance of the EPC molecules of the outer monolayer was shifted and broadened upon ACTH binding to the lipid vesicles, while that of the inner layer was not affected, suggesting that the peptide molecules interact only with the external leaflet of the lipid bilayer. The C²H and C⁴H resonances of the histidine-6 side chain were both shifted downfield upon peptide binding to the negatively charged lipid interface. In the case of the 1–24 derivative, these resonances were also split into two signals reflecting two different species of membrane-bound ACTH 1–24. Analysis of the line width and chemical shift variations of the ACTH and lipid resonances observed upon peptide binding shows that the membrane-binding potency of the shorter 5–14⁺¹ fragment, which presents a +1 net charge, is roughly similar to that of the highly cationic 1–24⁺⁶ (net charge +6) derivative, implying that the 15–24⁺⁵ segment is not essential for membrane binding. The nmr measurements at a fixed lipid-to-peptide ratio in the presence of increasing amounts of spin-labeled lipids demonstrate that the N-terminal fragment of ACTH does not penetrate the hydrophobic core of the bilayer, and should lie parallel to the membrane surface. © 1997 John Wiley & Sons, Inc. Biopoly 42: 731–744, 1997     

Effects of tryptophan residues of porcine myeloid antibacterial peptide PMAP-23 on antibiotic activity.

PMAP-23 is a 23-residue antimicrobial peptide from porcine myeloid cells. In order to determine the effects of two Trp residues in positions 7 and 21 of PMAP-23 on antibacterial activity and phospholipid vesicle interacting property, two analogues in which Ala is substituted for Trp residue in position 7 or 21 were synthesized. A(21)-PMAP-23 exhibited reduced antibacterial activity and phospholipid vesicle disrupting activity when compared to those of PMAP-23 and A(7)-PMAP-23. PMAP-23 readily interacted with model lipid membrane and induced membrane destabilization. Therefore antibacterial activity induced by PMAP-23 is due to the interaction of cell membrane with peptide followed by membrane perturbation. A significant structural change on the SDS micelle was not found by Ala substitution of the Trp residue of PMAP-23. Also, there is a good correlation between hydrophobic interaction on RP-HPLC, expressed as retention time on RP-HPLC, and antibacterial activity. The vesicle titration experiment indicated that Trp residues located at near C-terminus are accessible to hydrophobic tail of phospholipid vesicle. This result suggests that the C-terminal end of PMAP-23 penetrates into the lipid bilayer in the course of the interaction with phospholipid membranes and is important for its antibacterial activity.     

Investigation of the membrane-active peptides melittin and glucagon by photochemically induced dynamic-nuclear-polarization (photo-CIDNP) NMR

The photochemically induced dynamic-nuclear-polarization (photo-CIDNP) NMR technique was used to investigate the membrane-active peptides melittin and glucagon. The experiments were performed both in the absence and presence of phospholipid vesicles in order to study the topography of the membrane-bound state. From the results it can be concluded that the melittin peptide chain is oriented in such a way that the single tryptophan residue (Trp19) reaches into the membrane. In the case of glucagon, a binding interaction with vesicle membranes is indicated within the pH range 2-10, whereby the single tryptophan residue (Trp25) is buried in the lipid bilayer and the tyrosine and histidine residues are exposed to the aqueous solvent.     

Dimer structure of magainin 2 bound to phospholipid vesicles

Magainin 2 from African clawed frog Xenopus laevis is an antimicrobial peptide with broad spectra and action mechanisms considered to permeabilize bacterial membranes. CD, vibration, and solid-state NMR spectroscopies indicate the peptide adopts an alpha-helical conformation on binding to phospholipid bilayers, and its micelle-bound conformation, being monomeric and alpha-helical, is well detailed. We showed, however, that the peptide dimerizes on binding to phospholipid bilayers. This difference in the conformation and aggregation state between micelle- and bilayer-bound states prompted us to analyze the conformation of an equipotent analog of magainin 2 (F5Y,F16W magainin 2) bound to phosphatidylcholine vesicles using transferred nuclear Overhauser enhancement (TRNOE) spectroscopy. While observed medium-range TRNOE cross peaks were characteristic of alpha-helix, many long-range cross peaks were not compatible with the peptide's monomeric state. Simulated annealing calculations generated dimer structures indicating (1) two peptide molecules have a largely helical conformation in antiparallel orientation forming a short coiled-coil structure, (2) residues 4-20 are well converged and residues 9-20 are in an alpha-helical conformation, and (3) the interface of the two peptide molecules is formed by well-defined side chains of hydrophobic residues. Finally, determined structures are compatible with numerous investigations examining magainin-phospholipid interactions.     

Peptide-lipid interaction monitored by spin labeled biologically active melanocortin peptides

The present work comparatively analyzes the interaction of alpha-MSH and its more potent and long-acting analog [Nle4, D-Phe7]alpha-MSH (NDP-MSH) with lipid bilayers. The peptides were spin labeled with Toac (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at the N-terminal, as those derivatives had been previously shown to keep their full biological activity. Due to the special rigidity of the Toac covalent binding to the peptide molecule, this spin label is highly sensitive to the peptide backbone conformation and dynamics. The peptides were investigated both by the electron spin resonance (ESR) of Toac0 and the time resolved fluorescence of Trp9 present in the peptides. The Toac0 ESR of the membrane-bound peptides indicates that the two peptides are inserted into the bilayer, close to the bilayer surface, in rather similar environments. A residue titration around pKa 7.5, possibly that of His6, can be clearly monitored by peptide-lipid partition. Trp9 time resolved fluorescence indicates that the peptides, and their Toac-labeled derivatives, present rather similar conformations when membrane bound, though Trp9 in NDP-MSH, and in its Toac-labeled derivative, goes somewhat further down into the bilayer. Yet, Toac0 ESR signal shows that the Toac-labeled N-terminal of NDP-MSH is in a shallower position in the bilayer, as compared to the hormone.     

Isolation and characterization of a proteinase from the sarcocarp of melon fruit.

A proteinase from the sarcocarp of melon (Cucumis Melo L. var. Prince) was purified by a three-step procedure involving batch-wise treatment with CM-cellulose fibers, column chromatography on CM-cellulose powder and gel filtration on Sephadex G-75. The final enzyme preparation was homogeneous on acrylamide gel electrophoresis. Its molecular weight was estimated by two different methods to be about 50,000. Anlayses indicated tha presence of 475 amino acid residues and at least 7 moles of hexose. The maximum activity was found in the alkaline pH region against casein as a substrate. The optimum temperature against casein was 70 degrees at pH 7.1. The enzyme was strongly inhibited by diisopropyl fluorophosphate, partly inhibited by HgCl2 and not inhibited by EDTA, p-chloromercuribenzoic acid, N-tosyl-L-lysine chloromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone, and soybean trypsin inhibitor. The reduced and carboxymethylated insulin B-chain was cleaved at the peptide bonds of Asn3-Gln4, Cm-Cys7-Gly8, Glu13-Ala14, Leu15-Tyr16, Cm-Cys19-Gly20, Phe25-Tyr26, Pro28-Lys29, and Lys29-Ala30 by the enzyme.     

Interaction mechanism between microtubule-associated proteins and microtubules. A proton nuclear magnetic resonance analysis on the binding of synthetic peptide to tubulin

An amino acid sequence essential for microtubule-associated proteins (MAPs) to bind to microtubules is presented [Aizawa et al. (1989) J. Biol. Chem. 264, 5885-5890]. A synthetic peptide of 23 amino acid residues which corresponded to the sequence [tubulin binding peptide (TBP)] was active in binding to tubulin and inducing its assembly. The TBP-tubulin interaction mechanism was analyzed by proton nuclear magnetic resonance spectroscopy as a simplified model for MAP-microtubule interactions. Intraresidue transferred nuclear Overhauser effects (TRNOEs) of TBP in TBP-tubulin mixtures were analyzed, and strong binding of two Val and two Lys residues of TBP to tubulin was detected. Among the sharply peaked signals from tubulin aromatic residues, those due to Tyr ring protons broadened upon mixing with TBP, suggesting the involvement of Tyr residue(s) in the binding with TBP. Irradiation of the tubulin Tyr protons resulted in an intermolecular TRNOE at TBP methyl proton resonances. Evidently, hydrophobic interactions between Val and Tyr residues are important for the binding of TBP to tubulin. Hydrophobic interactions have not been taken into account previously in the widely accepted electrostatic model for the binding of MAPs to microtubules.     

Treatment of an encephalitogenic peptide from guinea pig myelin basic protein with alpha-protease and thermolysin. Isolation of fragments and determination of cleavage sites.

Two peptic fragments (residues 37-88 and 43-88) of guinea pig myelin basic protein which are capable of inducing experimental allergic encephalomyelitis in Lewis rats were cleaved to shorter fragments with alpha-protease (Crotalus atrox proteinase, EC 3.4.24.1) and thermolysin (EC 3.4.24.4). The fragments were isolated, purified, and identified by amino acid composition and NH2- and COOH-terminal residues. The time courses of the reactions, monitored by thin layer electrophoresis of the digests, showed that alpha-protease cleaves peptide (43-88) initially at the Pro(71)-Gln(72) bond, and that the product peptides are subsequently attacked at the Arg(63) -Thr(64), Ser(74)-Gln(75), Arg(78)-Ser(79), and Ser(76)-Gln(80) bonds. No significant cleavages occurred at the -Leu, -Val, and -Ala bonds. These results are in striking contrast to those obtained previously by others workers with other peptide substrates, where selective cleavage at hydrophobic residues occurred. Thermolysin was found to attack peptide (37-88) at the Phe(42)-Phe(43) bond very rapidly; the product peptides were subsequently attacked at the His(60)-Ala(61), Ser(38)-Ile(39)-Tyr(67)-Gly(68), and Pro(84)-Val(85) bonds. These cleavages are compatible with the known specificity of this enzyme. Several of the fragments prepared with these two enzymes, peptides (43-71), (61-88), (75-88), and (72-84) have been used in other studies to locate the encephalitogenic site in the parent peptic peptide.     

Solution structure of human apolipoprotein(a) kringle IV type 6.

The structure of apo(a) KIVT6 was investigated by two- and three-dimensional homo- and heteronuclear NMR spectroscopy. The solution structure of apo(a) KIVT6 contains only a small amount of regular secondary structure elements, comprising a short piece of antiparallel beta-sheet formed by residues Trp62-Tyr64 and Trp72-Tyr74, a short piece of parallel beta-sheet formed by the residues Cys1-Tyr2 and Thr78-Gln79, and a small 3(10)-helix within residues Thr38-Tyr40. The backbone as well as the side chains are arranged in a way similar to those of apo(a) KIVT7, apo(a) KIVT10, and plasminogen K4. We determined additionally the K(d) value of 0.31 +/- 0.04 mM for the binding of epsilon-aminocaproic acid (EACA) to apo(a) KIVT6 and mapped the binding region on apo(a) KIVT6 by means of chemical shift perturbation. This lysine binding activity, which was reported to occur within apo(a) KIVT5-8, is functionally different from the lysine binding activity found for apo(a) KIVT10.     

Molecular dynamics simulation of neuropeptide B and neuropeptide W in the dipalmitoylphosphatidylcholine membrane bilayer

GPR7 and GPR8 are recently deorphanized G-protein-coupled receptors that are implicated in the regulation of neuroendocrine function, feeding behavior, and energy homeostasis. Neuropeptide B (NPB) and neuropeptide W (NPW) are two membrane-bound hypothalamic peptides, which specifically antagonize GPR7 and GPR8. Despite years of research, an accurate estimation of structure and molecular recognition of these neuropeptide systems still remains elusive. Herein, we investigated the structure, orientation, and interaction of NPB and NPW in a dipalmitoylphosphatidylcholine bilayer using long-range molecular dynamics (MD) simulation. During 30-ns simulation, membrane-embedded helical axes of NPB and NPW tilted 30 and 15°, respectively, from the membrane normal in order to overcome possible hydrophobic mismatch with the lipid bilayer. The calculation of various structural parameters indicated that NPW is more rigid and compact as compared to NPB. Qualitatively, the peptides exhibited flexible N-terminal (residues 1–12) and rigid C-terminal α-helical parts (residues 13–21), confirming previous NMR data. A strong electrostatic attraction between C-termini and headgroup atoms caused translocation of the peptides towards lower leaflet of the bilayer. The stabilizing hydrogen bonds (H-bonds) between phosphate groups and Trp1, Lys3, and Arg15 of the peptides played important roles for membrane anchoring. MD simulations of Alanine (Ala) mutants revealed that WYK->Ala variant of NPB/NPW lacked crucial H-bond interactions with phospholipid headgroups and also caused severe misfolding in NPB. Altogether, the knowledge of preferred structural fold and interaction of neuropeptides within the membrane bilayer will be useful to develop synthetic agonist or antagonist peptides for GPR7 and GPR8.     

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 interaction of Escherichia coli hemolysin: flotation and insertion-dependent labeling by phospholipid vesicles

The 1,024-amino-acid acylated hemolysin of Escherichia coli subverts host cell functions and causes cell lysis. Both activities require insertion of the toxin into target mammalian cell membranes. To identify directly the principal toxin sequences dictating membrane binding and insertion, we assayed the lipid bilayer interaction of native protoxin, stably active toxin, and recombinant peptides. Binding was assessed by flotation of protein-liposome mixtures through density gradients, and insertion was assessed by labeling with a photoactivatable probe incorporated into the target lipid bilayer. Both the active acylated hemolysin and the inactive unacylated protoxin were able to bind and also insert. Ca(2+) binding, which is required for toxin activity, did not influence the in vitro interaction with liposomes. Three overlapping large peptides were expressed separately. A C-terminal peptide including residues 601 to 1024 did not interact in either assay. An internal peptide spanning residues 496 to 831, including the two acylation sites, bound to phospholipid vesicles and showed a low level of insertion-dependent labeling. In vitro acylation had no effect on the bilayer interaction of either this peptide or the full-length protoxin. An N-terminal peptide comprising residues 1 to 520 also bound to phospholipid vesicles and showed strong insertion-dependent labeling, ca. 5- to 25-fold that of the internal peptide. Generation of five smaller peptides from the N-terminal region identified the principal determinant of lipid insertion as the hydrophobic sequence encompassing residues 177 to 411, which is conserved among hemolysin-related toxins.     

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.