Conformational mapping of the N-terminal peptide of HIV-1 gp41 in membrane environments using (13)C-enhanced Fourier transform infrared spectroscopy

The N-terminal domain of HIV-1 glycoprotein 41000 (FP; residues 1--23; AVGIGALFLGFLGAAGSTMGARSCONH(2)) participates in fusion processes underlying virus--cell infection. Here, we use physical techniques to study the secondary conformation of synthetic FP in aqueous, structure-promoting, lipid and biomembrane environments. Circular dichroism and conventional, (12)C-Fourier transform infrared (FTIR) spectroscopy indicated the following alpha-helical levels for FP in 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) liposomes-hexafluoroisopropanol (HFIP)>trifluoroethanol (TFE)>phosphate-buffered saline (PBS). (12)C-FTIR spectra also showed disordered FP structures in these environments, along with substantial beta-structures for FP in TFE or PBS. In further experiments designed to map secondary conformations to specific residues, isotope-enhanced FTIR spectroscopy was performed using a suite of FP peptides labeled with (13)C-carbonyl at multiple sites. Combining these (13)C-enhanced FTIR results with molecular simulations indicated the following model for FP in HFIP: alpha-helix (residues 3-16) and random and beta-structures (residues 1-2 and residues 17-23). Additional (13)C-FTIR analysis indicated a similar conformation for FP in POPG at low peptide loading, except that the alpha-helix extends over residues 1-16. At low peptide loading in either human erythrocyte ghosts or lipid extracts from ghosts, (13)C-FTIR spectroscopy showed alpha-helical conformations for the central core of FP (residues 5-15); on the other hand, at high peptide loading in ghosts or lipid extracts, the central core of FP assumed an antiparallel beta-structure. FP at low loading in ghosts probably inserts deeply as an alpha-helix into the hydrophobic membrane bilayer, while at higher loading FP primarily associates with ghosts as an aqueous-accessible, beta-sheet. In future studies, (13)C-FTIR spectroscopy may yield residue-specific conformations for other membrane-bound proteins or peptides, which have been difficult to analyze with more standard methodologies.     

Conformational mapping of the N-terminal peptide of HIV-1 gp41 in membrane environments using C-enhanced Fourier transform infrared spectroscopy

The N-terminal domain of HIV-1 glycoprotein 41?000 (FP; residues 1-23; AVGIGALFLGFLGAAGSTMGARSCONH₂) participates in fusion processes underlying virus-cell infection. Here, we use physical techniques to study the secondary conformation of synthetic FP in aqueous, structure-promoting, lipid and biomembrane environments. Circular dichroism and conventional, ¹²C-Fourier transform infrared (FTIR) spectroscopy indicated the following α-helical levels for FP in 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) liposomes∼hexafluoroisopropanol (HFIP)>trifluoroethanol (TFE)>phosphate-buffered saline (PBS). ¹²C-FTIR spectra also showed disordered FP structures in these environments, along with substantial β-structures for FP in TFE or PBS. In further experiments designed to map secondary conformations to specific residues, isotope-enhanced FTIR spectroscopy was performed using a suite of FP peptides labeled with ¹³C-carbonyl at multiple sites. Combining these ¹³C-enhanced FTIR results with molecular simulations indicated the following model for FP in HFIP: α-helix (residues 3-16) and random and β-structures (residues 1-2 and residues 17-23). Additional ¹³C-FTIR analysis indicated a similar conformation for FP in POPG at low peptide loading, except that the α-helix extends over residues 1-16. At low peptide loading in either human erythrocyte ghosts or lipid extracts from ghosts, ¹³C-FTIR spectroscopy showed α-helical conformations for the central core of FP (residues 5-15); on the other hand, at high peptide loading in ghosts or lipid extracts, the central core of FP assumed an antiparallel β-structure. FP at low loading in ghosts probably inserts deeply as an α-helix into the hydrophobic membrane bilayer, while at higher loading FP primarily associates with ghosts as an aqueous-accessible, β-sheet. In future studies, ¹³C-FTIR spectroscopy may yield residue-specific conformations for other membrane-bound proteins or peptides, which have been difficult to analyze with more standard methodologies.     

Conformation and molecular topography of the N-terminal segment of surfactant protein B in structure-promoting environments.

Although the effects of surfactant protein B (SP-B) on lipid surface activity in vitro and in vivo are well known, the relationship between molecular structure and function is still not fully understood. To further characterize protein structure-activity correlations, we have used physical techniques to study conformation, orientation, and molecular topography of N-terminal SP-B peptides in lipids and structure-promoting environments. Fourier transform infrared (FTIR) and CD measurements of SP-B1-25 (residues 1-25) in methanol, SDS micelles, egg yolk lecithin (EYL) liposomes, and surfactant lipids indicate the peptide has a dominant helical content, with minor turn and disordered components. Polarized FTIR studies of SP-B1-25 indicate the long molecular axis lies at an oblique angle to the surface of lipid films. Truncated peptides were similarly examined to assign more accurately the discrete conformations within the SP-B1-25 sequence. Residues Cys-8-Gly-25 are largely alpha-helix in methanol, whereas the N-terminal segment Phe-1-Cys-8 had turn and helical propensities. Addition of SP-B1-25 spin-labeled at the N-terminal Phe (i.e., SP-B1-25) to SDS, EYL, or surfactant lipids yielded electron spin resonance spectra that reflect peptide bound to lipids, but retaining considerable mobility. The absence of characteristic radical broadening indicates that SP-B1-25 is minimally aggregated when it interacts with these lipids. Further, the high polarity of SP-B1-25 argues that the reporter on Phe-1 resides in the headgroup of the lipid dispersions. The blue-shift in the endogenous fluorescence of Trp-9 near the N-terminus of SP-B1-25 suggests that this residue also lies near the lipid headgroup. A summary model based on the above physical experiments is presented for SP-B1-25 interacting with lipids.     

Conformational mapping of the N-terminal peptide of HIV-1 gp41 in lipid detergent and aqueous environments using 13C-enhanced Fourier transform infrared spectroscopy

The N-terminal domain of HIV-1 glycoprotein 41,000 (gp41) participates in viral fusion processes. Here, we use physical and computational methodologies to examine the secondary structure of a peptide based on the N terminus (FP; residues 1-23) in aqueous and detergent environments. (12)C-Fourier transform infrared (FTIR) spectroscopy indicated greater alpha-helix for FP in lipid-detergent sodium dodecyl sulfate (SDS) and aqueous phosphate-buffered saline (PBS) than in only PBS. (12)C-FTIR spectra also showed disordered FP conformations in these two environments, along with substantial beta-structure for FP alone in PBS. In experiments that map conformations to specific residues, isotope-enhanced FTIR spectroscopy was performed using FP peptides labeled with (13)C-carbonyl. (13)C-FTIR results on FP in SDS at low peptide loading indicated alpha-helix (residues 5 to 16) and disordered conformations (residues 1-4). Because earlier (13)C-FTIR analysis of FP in lipid bilayers demonstrated alpha-helix for residues 1-16 at low peptide loading, the FP structure in SDS micelles only approximates that found for FP with membranes. Molecular dynamics simulations of FP in an explicit SDS micelle indicate that the fraying of the first three to four residues may be due to the FP helix moving to one end of the micelle. In PBS alone, however, electron microscopy of FP showed large fibrils, while (13)C-FTIR spectra demonstrated antiparallel beta-sheet for FP (residues 1-12), analogous to that reported for amyloid peptides. Because FP and amyloid peptides each exhibit plaque formation, alpha-helix to beta-sheet interconversion, and membrane fusion activity, amyloid and N-terminal gp41 peptides may belong to the same superfamily of proteins.     

Lipid bilayer surface association of lung surfactant protein SP-B, amphipathic segment detected by flow immunofluorescence.

Lung surfactant protein, SP-B, and synthetic amphipathic peptides derived from SP-B were studied in model lung surfactant lipid bilayers by immunofluorescent labeling. Liposomes were formed by hydrating a lipid film on the glass viewing port of a temperature controlled flow chamber. Membrane associated peptides were detected by epifluorescence optical microscopy of the binding of anti-peptide polyclonal monospecific antibodies and FITC-conjugated secondary antibodies added to buffer contained in the flow chamber. Liposomes were bound by antibody to residues 1-25 of SP-B if formed from lipid films containing the 1-25 peptide, (SP-B(1-25)), or if SP-B(1-25) was added to already formed liposomes in buffer solution. The distribution of antigen-antibody complex was temperature dependent with aggregation occurring at greater than or equal to 30 degrees C. Surface association was not detected in liposomes formed from lipid films containing the 49-66 peptides (SP-B(49-66)), using an antibody to the 49-66 peptide, or to a synthetic version of the SP-B protein, (SP-B(1-78)), using both antibodies to the 49-66 peptide and the 1-25 peptide. The detection of SP-B(1-78) with antibody to the 49-66 sequence was only possible after reducing SP-B(1-78) with dithiothreitol, suggesting that the COOH-terminus of the full monomer protein is accessible to the bulk aqueous environment unlike the COOH-terminal peptide. The size, number of layers, and fluidity of the liposomes were not altered by protein or peptides, although they were affected by lipid composition and temperature.     

Multilayer formation upon compression of surfactant monolayers depends on protein concentration as well as lipid composition. An atomic force microscopy study

The determinants for the formation of multilayers upon compression of surfactant monolayers were investigated by compressing films, beyond the squeeze-out plateau, to a surface tension of 22 millinewtons/m. Atomic force microscopy was used to visualize the topography of lipid films containing varying amounts of native surfactant protein B (SP-B). These films were compared with films containing synthetic peptides based on the N terminus of human SP-B: monomeric mSP-B-(1-25) or dimeric dSP-B-(1-25). The formation of typical hexagonal network structures as well as the height of protrusions were shown to depend on the concentration of SP-B. Protrusions of bilayer height were formed from physiologically relevant concentrations of 0.2-0.4 mol % (4.5-8.5 wt %) SP-B upwards. Much higher concentrations of SP-B-(1-25) peptides were needed to obtain network structures, and protrusion heights were not equal to those found for films with native SP-B. A striking observation was that while protrusions formed in films of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dipalmitoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (DPPG) (80/20) had single bilayer thickness, those formed in DPPC/1-palmitoyl-2-oleoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (80/20) had various heights of multilayers, whereas those seen in DPPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/DPPG (60/20/20) were mainly of bilayer height. For the first time direct observations by atomic force microscopy show (i) that a certain minimal concentration of SP-B is required for the formation of layered protrusions upon film compression, (ii) that protrusion height depends on whether the phospholipids contain an unsaturated fatty acyl chain, and (iii) that protrusion height also depends on whether the unsaturated acyl chain is present in phosphatidylcholine or in phosphatidylglycerol.     

Effects of surfactant-associated protein SP-B synthetic analogs on the structure and surface activity of model membrane bilayers.

The effect of several synthetic peptides based on the sequence of human pulmonary surfactant-associated protein B (SPB) on the molecular packing of model membrane lipids (7:1 dipalmitoyl phosphatidylcholine (DPPC)/dipalmitoyl phosphatidylglycerol (DPPG)) was studied using fluorescence anisotropy. This information was then correlated with complementary biophysical data obtained on both a modified Wilhelmy-Langmuir balance and a pulsating bubble surfactometer. The SP-B peptides examined in these studies are synthetic human SP-B Phe1-Ser78 (SP-B 1-78, full-length sequence), synthetic human SP-B Phe1-Thr60 (SP-B 1-60), synthetic human SP-B Phe1-Ala20 (SP-B 1-20), synthetic human SP-B Ala20-Thr60 (SP-B 20-60), synthetic human SP-B Leu27-Ser78 (SP-B 27-78), synthetic human SP-B Leu40-Thr60 (SP-B 40-60) and synthetic human SP-B Tyr53-Ser78 (SP-B 53-78). trans-parinaric acid was utilized to detect changes in ordering of lipids within the interior upon incorporation of synthetic SP-B peptide, whereas 1-hexadecanoyl-2-[N-(7-nitro-2-benzoxa-1,3-diazol-4-yl)-a min ohexanoyl] phosphatidylcholine (6-NBD-PC) and 1-acyl-2-[N-(7-nitro-2-benzoxa-1,3-diazol-4-yl)aminohexanoyl ] phosphatidylglycerol (6-NBD-PG) were utilized to determine alterations in lipid order at the surface of the model membrane bilayer. With the exception of SP-B 40-60, which corresponds to the most hydrophobic segment of the full-length SP-B, none of the other peptide significantly perturbed the interior bilayer as determined by fluorescence anisotropy of trans-parinaric acid. Incorporation of any of the peptides with the exception of SP-B 40-60, resulted in an increase in anisotropy of NBD-PC. The most significant enhancements resulted from the addition of SP-B 1-78, SP-B 1-20, SP-B 27-78 or SP-B 53-78. The magnitude of anisotropy increase with these peptides is similar to that observed with an equivalent molar ratio of native SP-B isolated from a bovine source. These observations suggest that these four synthetic peptides have the structural and compositional characteristics required for surface ordering of the membrane bilayer in a manner similar to that observed with native SP-B, thereby facilitating the surfactant-like properties of phospholipid mixtures.     

Mapping and analysis of the lytic and fusogenic domains of surfactant protein B

Surfactant protein B (SP-B) is a hydrophobic, 79 amino acid peptide that regulates the structure and function of surfactant phospholipid membranes in the airspaces of the lung. Addition of SP-B to liposomes composed of DPPC/PG (7:3) leads to membrane binding, destabilization, and fusion, ultimately resulting in rearrangement of membrane structure. The goal of this study was to map the fusogenic and lytic domains of SP-B and assess the effects of altered fusion and lysis on surface activity. Synthetic peptides were generated to predicted helices and/or interhelical loops of SP-B and tested for fusion, lytic, and surface activities. The N-terminal half of SP-B (residues 1-37), which includes the nonhelical N-terminal amino acids in addition to helices 1 and 2, promoted rapid liposome fusion whereas shorter peptides were significantly less effective. The requirements for optimal surface tension reduction were similar to those for fusion; in contrast, helix 1 (residues 7-22) alone was sufficient for liposome lysis. The C-terminal half of SP-B (residues 43-79), which includes helices 3, 4, and 5, exhibited significantly lower levels of fusogenic, lytic, and surface tension reducing activities compared to the N-terminal region. These results indicate that SP-B fusion, lytic and surface activities map predominantly to the N-terminal half of SP-B. Amino acid substitutions in synthetic peptides corresponding to the N-terminal half of SP-B indicated that, in general, decreased fusion or lytic activities were associated with altered surface tension reducing properties of the peptide. However, the presence of fusion and lytic activities alone could not account for the surface tension reducing property of SP-B. We propose a model in which association of helix 1 with lipids leads to membrane permeabilization but not aggregation; helix 2 mediates membrane cross-linking (aggregation), which, in turn, facilitates lipid mixing, membrane fusion, and interfacial adsorption/surface tension reduction.     

Membrane structure and interactions of peptide hormones with model lipid bilayers

In this work, the behavior of the neurohypophyseal hormones and their selected analogs was studied in the presence of membrane models in an attempt to correlate their activities with a distinct behavior at a level of peptide-lipid interactions. The influence of the peptides studied on the lipid acyl chain order was determined using FTIR spectroscopy. Conformational changes in the peptides upon binding to liposomes were examined using CD spectra. Attempts were also made to determine the binding parameters of the peptides to lipids using isothermal titration calorimetry (ITC). The results show unambiguously that the neurohyphophyseal hormone-like peptides interact with lipids, being a model of a eukaryotic cell membrane. Moreover, hydrophobic interactions between the peptides and liposomes are likely to determine the overall conformation of the peptide, especially below the temperature of the main phase transition (T(m)). Thus, the bulky and hydrophobic nature of the residues incorporated into the N-terminal part of neurohyphophyseal hormones is an important factor for both restriction of peptide mobility and the interaction of the analog with biomembrane. In turn, above T(m), the electrostatic interactions become also relevant for the conformation of the acyclic tail of the AVP-like peptides.     

Raman spectroscopic studies of model human pulmonary surfactant systems: phospholipid interactions with peptide paradigms for the surfactant protein SP-B

The temperature dependence of dipalmitoylphosphatidylcholine (DPPC)/phosphatidylglycerol (PG) multilayers, reconstituted with various synthetic peptides for modeling human lung surfactant, was monitored by vibrational Raman spectroscopy. The synthetic peptides consisted, respectively, of residues 59-81 of the human surfactant protein SP-B and 21 amino acid residue peptides containing repeating units of arginine separated by either four or eight leucines (RL4 or RL8). Each peptide demonstrated the ability to reduce significantly the surface tension of analogues of the phospholipid mixture used in the Raman studies. Raman spectroscopic integrated band intensities and relative peak height intensity ratios, two spectral parameters used to determine bilayer disorder, provided sensitive probes for characterizing multilayer perturbations in the reconstituted liposomes. Temperature profiles derived from the various Raman intensity parameters for the 3100-2800-cm-1 carbon-hydrogen (C-H) stretching mode region, a spectral interval representative of acyl chain vibrations, reflected lipid reorganizations due to the bilayer interactions of these peptides. For the three reconstituted multilamellar surfactant systems, the gel-to-liquid-crystalline phase-transition temperatures Tm, defined by acyl chain C-H stretching mode order/disorder parameters, increased from 35 degrees C in the peptide free system to 37-38 degrees C, indicating increased lipid headgroup constraints for the model liposomes. Although the values of Tm were similar for the three recombinant lipid/peptide assemblies, individual phase-transition cooperativities varied significantly between systems and between spectroscopically derived order/disorder parameters.     

Structure and conformation of the disulfide bond in dimeric lung surfactant peptides SP-B1-25 and SP-B8-25

Raman spectroscopy was used to determine the conformation of the disulfide linkage between cysteine residues in the homodimeric construct of the N-terminal alpha helical domain of surfactant protein B (dSP-B(1-25)). The conformation of the disulfide bond between cysteine residues in position 8 of the homodimer of dSP-B(1-25) was compared with that of a truncated homodimer (dSP-B(8-25)) of the peptide having a disulfide linkage at the same position in the alpha helix. Temperature-dependent Raman spectra of the S-S stretching region centered at approximately 500 cm(-1) indicated a stable, although highly strained disulfide conformation with a chi(CS-SC) dihedral angle of +/-10 degrees for the dSP-B(1-25) dimer. In contrast, the truncated dimer dSP-B(8-25) exhibited a series of disulfide conformations with the chi(CS-SC) dihedral angle taking on values of either +/-30 degrees or 85+/-20 degrees . For conformations with chi(CS-SC) close to the +/-90 degrees value, the Raman spectra of the 8-25 truncated dimers exhibited chi(SS-CC) dihedral angles of 90/180 degrees and 20-30 degrees . In the presence of a lipid mixture, both constructs showed a nu(S-S) band at approximately 488 cm(-1), corresponding to a chi(CS-SC) dihedral angle of +/-10 degrees . Polarized infrared spectroscopy was also used to determine the orientation of the helix and beta-sheet portion of both synthetic peptides. These calculations indicated that the helix was oriented primarily in the plane of the surface, at an angle of approximately 60-70 degrees to the surface normal, while the beta structure had approximately 40 degrees tilt. This orientation direction did not change in the presence of a lipid mixture or with temperature. These observations suggest that: (i) the conformational flexibility of the disulfide linkage is dependent on the amino acid residues that flank the cysteine disulfide bond, and (ii) in both constructs, the presence of a lipid matrix locks the disulfide bond into a preferred conformation.     

Critical Structural and Functional Roles for the N-Terminal Insertion Sequence in Surfactant Protein B Analogs

Background

Surfactant protein B (SP-B; 79 residues) belongs to the saposin protein superfamily, and plays functional roles in lung surfactant. The disulfide cross-linked, N- and C-terminal domains of SP-B have been theoretically predicted to fold as charged, amphipathic helices, suggesting their participation in surfactant activities. Earlier structural studies with Mini-B, a disulfide-linked construct based on the N- and C-terminal regions of SP-B (i.e., ∼residues 8–25 and 63–78), confirmed that these neighboring domains are helical; moreover, Mini-B retains critical in vitro and in vivo surfactant functions of the native protein. Here, we perform similar analyses on a Super Mini-B construct that has native SP-B residues (1–7) attached to the N-terminus of Mini-B, to test whether the N-terminal sequence is also involved in surfactant activity.

Methodology/Results

FTIR spectra of Mini-B and Super Mini-B in either lipids or lipid-mimics indicated that these peptides share similar conformations, with primary α-helix and secondary β-sheet and loop-turns. Gel electrophoresis demonstrated that Super Mini-B was dimeric in SDS detergent-polyacrylamide, while Mini-B was monomeric. Surface plasmon resonance (SPR), predictive aggregation algorithms, and molecular dynamics (MD) and docking simulations further suggested a preliminary model for dimeric Super Mini-B, in which monomers self-associate to form a dimer peptide with a “saposin-like” fold. Similar to native SP-B, both Mini-B and Super Mini-B exhibit in vitro activity with spread films showing near-zero minimum surface tension during cycling using captive bubble surfactometry. In vivo, Super Mini-B demonstrates oxygenation and dynamic compliance that are greater than Mini-B and compare favorably to full-length SP-B.

Conclusion

Super Mini-B shows enhanced surfactant activity, probably due to the self-assembly of monomer peptide into dimer Super Mini-B that mimics the functions and putative structure of native SP-B.     

NMR study of a 34-residue N-terminal fragment of the parathyroid-hormone-related protein secreted during humoral hypercalcemia of malignancy

The proton resonances of the biologically active peptide parathyroid-hormone-related protein (residues 1-34) were assigned using one-dimensional spin-decoupling techniques, two-dimensional correlated spectroscopy and by comparing the spectra of the peptides 1-20, 1-25, 1-29, 7-34 and 15-34. The conformation of 1-34 was determined using one- and two-dimensional nuclear Overhauser enhancement spectroscopy in the rotating frame. Amide proton temperature coefficients, vicinal coupling constants and circular dichroic spectra helped reveal a surprisingly compact structure with residues 3-9 forming alpha-helix, type-I beta-turns between residues 10-13 and 16-19 and several interactions between the N-terminal residues and the C-terminal residues. Of these latter, the strongest appeared to be between Asp-10 and Phe-22. One peptide surface in the deduced model presents multiple positive charges, while the opposite surface has a hydrophobic character possibly functioning to exclude water from the binding interface and enhancing the binding constant.     

Relative spatial positions of tryptophan and cationic residues in helical membrane-active peptides determine their cytotoxicity

The cytotoxic activity of 10 analogs of the idealized amphipathic helical 21-mer peptide (KAAKKAA)3, where three of the Ala residues at different positions have been replaced with Trp residues, has been investigated. The peptide's cytotoxic activity was found to be markedly dependent upon the position of the Trp residues within the hydrophobic sector of an idealized α-helix. The peptides with Trp residues located opposite the cationic sector displayed no antitumor activity, whereas those peptides with two or three Trp residues located adjacent to the cationic sector exhibited high cytotoxic activity when tested against three different cancer cell lines. Dye release experiments revealed that in contrast to the peptides with Trp residues located opposite the cationic sector, the peptides with Trp residues located adjacent to the cationic sector induced a strong permeabilizing activity from liposomes composed of a mixture of zwitterionic phosphatidylcholine and negatively charged phosphatidylserine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS)) (2:1) but not from liposomes composed of zwitterionic phosphatidylcholine, POPC. Fluorescence blue shift and quenching experiments revealed that Trp residues inserted deeper into the hydrophobic environment of POPC/POPS liposomes for peptides with high cytotoxic activity. Through circular dichroism studies, a correlation between the cytotoxic activity and the α-helical propensity was established. Structural studies of one inactive and two active peptides in the presence of micelles using NMR spectroscopy showed that only the active peptides adopted highly coiled to helical structures when bound to a membrane surface.     

Rationale for the design of shortened derivatives of the NK-lysin-derived antimicrobial peptide NK-2 with improved activity against Gram-negative pathogens

The peptide NK-2 is an effective antimicrobial agent with low hemolytic and cytotoxic activities and is thus a promising candidate for clinical applications. It comprises the alpha-helical, cationic core region of porcine NK-lysin a homolog of human granulysin and of amoebapores of pathogenic amoeba. Here we visualized the impact of NK-2 on Escherichia coli by electron microscopy and used NK-2 as a template for sequence variations to improve the peptide stability and activity and to gain insight into the structure/function relationships. We synthesized 18 new peptides and tested their activities on seven Gram-negative and one Gram-positive bacterial strains, human erythrocytes, and HeLa cells. Although all peptides appeared unordered in buffer, those active against bacteria adopted an alpha-helical conformation in membrane-mimetic environments like trifluoroethanol and negatively charged phosphatidylglycerol (PG) liposomes that mimick the cytoplasmic membrane of bacteria. This conformation was not observed in the presence of liposomes consisting of zwitterionic phosphatidylcholine (PC) typical for the human cell plasma membrane. The interaction was paralleled by intercalation of these peptides into PG liposomes as determined by FRET spectroscopy. A comparative analysis between biological activity and the calculated peptide parameters revealed that the decisive factor for a broad spectrum activity is not the peptide overall hydrophobicity or amphipathicity, but the possession of a minimal positive net charge plus a highly amphipathic anchor point of only seven amino acid residues (two helical turns).     

Secondary structure and orientation of the surfactant protein SP-B in a lipid environment. A Fourier transform infrared spectroscopy study.

Attenuated total reflection Fourier transform infrared spectroscopy was used to investigate the secondary structure of the surfactant protein SP-B. Nearly half of the polypeptide chain is folded in an alpha-helical conformation. No significant change of the secondary structure content was observed when the protein is associated to a lipid bilayer of dipalmitoylphosphatidylcholine (DPPC)/phosphatidylglycerol (PG) or of dipalmitoylphosphatidylglycerol (DPPG). The parameters related to the gamma w(CH2) vibration of the saturated acyl chains reveal no modification of the conformation or orientation of the lipids in the presence of SP-B. A model of orientation of the protein at the lipid/water interface is proposed. In this model, electrostatic interactions between charged residues of SP-B and polar headgroups of PG, and the presence of small hydrophobic alpha-helical peptide stretches slightly inside the bilayers, would maintain SP-B at the membrane surface.     

Molecular dynamics simulations of the anchoring and tilting of the lung-surfactant peptide SP-B1-25 in palmitic acid monolayers

We have performed molecular dynamics simulations of multiple copies of the lung-surfactant peptide SP-B1-25 in a palmitic acid (PA) monolayer. SP-B1-25 is a shorter version of lung-surfactant protein B, an important component of lung surfactant. Up to 30 ns simulations of 20 wt % SP-B1-25 in the PA monolayers were performed with different surface areas of PA, extents of PA ionization, and various initial configurations of the peptides. Starting with initial peptide orientation perpendicular to the monolayer, the predicted final tilt angles average 54 degrees approximately 62 degrees with respect to the monolayer normal, similar to those measured experimentally by Lee et al. (Biophysical Journal. 2001. Synchrotron x-ray study of lung surfactant-specific protein SP-B in lipid monolayers. 81:572-585). In their final conformations, hydrogen-bond analysis and amino acid mutation studies show that the peptides are anchored by hydrogen bond interactions between the cationic residues Arg-12 and Arg-17 and the hydrogen bond acceptors of the ionized PA headgroup, and the tilt angle is affected by the interactions of Tyr-7 and Gln-19 with the PA headgroup. Our work indicates that the factors controlling orientation of small peptides in lipid layers can now be uncovered through molecular dynamics simulations.     

Interaction of synthetic fragments of the extension peptide of cytochrome P-450(SCC) precursor with phospholipid bilayer

Six peptide fragments, SEP6-11, SEP6-15, SEP1-11, SEP1-15, SEP1-20, and SEP25-39, corresponding to the residues 6-11, 6-15, 1-11, 1-15, 1-20, and 25-39, respectively, of the extension peptide of cytochrome P-450(SCC) precursor were chemically synthesized by the solution method. CD spectra of the peptides indicated that SEP1-15 and SEP1-20, which inhibit the import of the precursor, held a random conformation in a Tris-HCl buffer and a partially ordered conformation (like alpha-helix or type II' beta-turn) in a buffer containing acidic liposomes. Slightly changed spectra were observed for SEP1-15 and SEP1-20 in the buffer containing neutral liposomes and in MeOH, respectively. Other peptides which show weak or no inhibitory effect had almost random conformations in these solvents. The hydrophobic moments of SEP1-15 and SEP1-20 when they take alpha-helical conformation are very small, suggesting that amphiphilic helical property is not always necessary for the import of the precursor, although partially ordered conformation seems to be required. The ability of SEP1-15 and SEP1-20 to induce leakage of carboxyfluorescein from the inside of dipalmitoyl-DL-alpha-phosphatidylcholine (DPPC) or DPPC-dipalmitoyl-DL-alpha-phosphatidylglycerol (3:1) vesicles was much greater than that of other peptides. The leakage induction ability of the peptides qualitatively parallels the degree of their inhibition of the import of the precursor. Perturbation of the membranes caused by the action of the peptides in their partially ordered form may be important for the import of the precursor into mitochondria.     

Topographical organization of the N-terminal segment of lung pulmonary surfactant protein B (SP-B(1-25)) in phospholipid bilayers

The location and depth of each residue of lung pulmonary surfactant protein B (SP-B(1-25)) in a phospholipid bilayer (PB) was determined by fluorescence quenching using synthesized single-residue-substituted peptides that were reconstituted into 1,2-dipalmitoyl phosphatidylcholine (DPPC)-enriched liposomes. The single-residue substitutions in peptides were either aspartate or tryptophan. The aspartate was subsequently labeled with the N-cyclohexyl-N'-(4-(dimethylamino)naphthyl)carbodiimide (NCD-4) fluorophore, whereas tryptophan is autofluorescent. Spin-labeled compounds, 5-doxylstearic acid (5-DSA), 7-doxylstearic acid (7-DSA), 12-doxylstearic acid (12-DSA), 4-(N,N-dimethyl-N-hexadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-oxyl iodide (CAT-16), and 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-1-oxy iodide (CAT-1), were used in the quenching experiments. The effective quenching order is determined by the accessibility of the quencher to a fluorescent group on the peptide. The order of quenching efficiency provides information about the relative locations of individual residues in the PB. Our data indicate that residues Phe1-Pro6 are located at the surface of PB, residues Tyr7-Trp9 are embedded in PB, and residues Leu10-Ile22 are involved in an amphipathic alpha-helix with its axis parallel to the surface of PB; residues Pro23-Gly25 reside at the surface. The effects of intermolecular disulfide bond formation in the SP-B(1-25) dimer were also investigated. The experiments suggest that the SP-B helix A has to rotate at an angle to form a disulfide bond with the neighboring cysteine, which makes the hydrophobic sides of the amphipathic helices face each other, thus forming a hydrophobic domain. The detailed topographical mapping of SP-B(1-25) and its dimer in PB provides new insights into the conformational organization of the lung pulmonary surfactant proteins in the environment that mimics the native state. The environment-specific conformational flexibility of the hydrophobic domain created by SP-B folding may explain the key functional properties of SP-B including their impact on phospholipid transport between the lipid phases and in modulating the cell inflammatory response during respiratory distress syndrome.     

A 1H-NMR study of the solution conformation of Icaria chemotactic peptide and its [Lys7] analog: effects on the physiological activity of a substitution of proline to lysine at position 7

Solution conformations of Icaria chemotactic peptide isolated from the Ropalidian wasp venom and its [Lys7] analog have been analyzed by the use of two-dimensional proton nuclear resonance spectroscopy. It has been shown that Icaria chemotactic peptide takes alpha helical conformation in the C-terminal 8-13 segment with the N-terminal 1-7 segment disordered. By contrast, the [Lys7] analog takes alpha helical conformation over a wider range from residues 3 to 13. The present results indicate that the substitution of proline to lysine at position 7 results in a drastic change in solution conformation, providing the Icaria chemotactic peptide with new physiological functions.