Partitioning, dynamics, and orientation of lung surfactant peptide KL4 in phospholipid bilayers

Lung surfactant protein B (SP-B) is a lipophilic protein critical to lung function at ambient pressure. KL₄ is a 21-residue peptide which has successfully replaced SP-B in clinical trials of synthetic lung surfactants. CD and FTIR measurements indicate KL₄ is helical in a lipid bilayer environment, but its exact secondary structure and orientation within the bilayer remain controversial. To investigate the partitioning and dynamics of KL₄ in phospholipid bilayers, we introduced CD₃-enriched leucines at four positions along the peptide to serve as probes of side chain dynamics via ²H solid-state NMR. The chosen labels allow distinction between models of helical secondary structure as well as between a transmembrane orientation or partitioning in the plane of the lipid leaflets. Leucine side chains are also sensitive to helix packing interactions in peptides that oligomerize. The partitioning and orientation of KL₄ in DPPC/POPG and POPC/POPG phospholipid bilayers, as inferred from the leucine side chain dynamics, is consistent with monomeric KL₄ lying in the plane of the bilayers and adopting an unusual helical structure which confers amphipathicity and allows partitioning into the lipid hydrophobic interior. At physiologic temperatures, the partitioning depth and dynamics of the peptide are dependent on the degree of saturation present in the lipids. The deeper partitioning of KL₄ relative to antimicrobial amphipathic α-helices leads to negative membrane curvature strain as evidenced by the formation of hexagonal phase structures in a POPE/POPG phospholipid mixture on addition of KL₄. The unusual secondary structure of KL₄ and its ability to differentially partition into lipid lamellae containing varying levels of saturation suggest a mechanism for its role in restoring lung compliance.     

KL4 Peptide Induces Reversible Collapse Structures on Multiple Length Scales in Model Lung Surfactant

We investigated the effects of KL₄, a 21-residue amphipathic peptide approximating the overall ratio of positively charged to hydrophobic amino acids in surfactant protein B (SP-B), on the structure and collapse of dipalmitoylphosphatidylcholine and palmitoyl-oleoyl-phosphatidylglycerol monolayers. As reported in prior work on model lung surfactant phospholipid films containing SP-B and SP-B peptides, our experiments show that KL₄ improves surfactant film reversibility during repetitive interfacial cycling in association with the formation of reversible collapse structures on multiple length scales. Emphasis is on exploring a general mechanistic connection between peptide-induced nano- and microscale reversible collapse structures (silos and folds).     

KL₄ peptide induces reversible collapse structures on multiple length scales in model lung surfactant

We investigated the effects of KL₄, a 21-residue amphipathic peptide approximating the overall ratio of positively charged to hydrophobic amino acids in surfactant protein B (SP-B), on the structure and collapse of dipalmitoylphosphatidylcholine and palmitoyl-oleoyl-phosphatidylglycerol monolayers. As reported in prior work on model lung surfactant phospholipid films containing SP-B and SP-B peptides, our experiments show that KL₄ improves surfactant film reversibility during repetitive interfacial cycling in association with the formation of reversible collapse structures on multiple length scales. Emphasis is on exploring a general mechanistic connection between peptide-induced nano- and microscale reversible collapse structures (silos and folds).     

Penetration Depth of Surfactant Peptide KL4 into Membranes Is Determined by Fatty Acid Saturation

KL₄ is a 21-residue functional peptide mimic of lung surfactant protein B, an essential protein for lowering surface tension in the alveoli. Its ability to modify lipid properties and restore lung compliance was investigated with circular dichroism, differential scanning calorimetry, and solid-state NMR spectroscopy. KL₄ binds fluid lamellar phase PC/PG lipid membranes and forms an amphipathic helix that alters lipid organization and acyl chain dynamics. The binding and helicity of KL₄ is dependent on the level of monounsaturation in the fatty acid chains. At physiologic temperatures, KL₄ is more peripheral and dynamic in fluid phase POPC/POPG MLVs but is deeply inserted into fluid phase DPPC/POPG vesicles, resulting in immobilization of the peptide. Substantial increases in the acyl chain order are observed in DPPC/POPG lipid vesicles with increasing levels of KL₄, and POPC/POPG lipid vesicles show small decreases in the acyl chain order parameters on addition of KL₄. Additionally, a clear effect of KL₄ on the orientation of the fluid phase PG headgroups is observed, with similar changes in both lipid environments. Near the phase transition temperature of the DPPC/POPG lipid mixtures, which is just below the physiologic temperature of lung surfactant, KL₄ causes phase separation with the DPPC remaining in a gel phase and the POPG partitioned between gel and fluid phases. The ability of KL₄ to differentially partition into lipid lamellae containing varying levels of monounsaturation and subsequent changes in curvature strain suggest a mechanism for peptide-mediated lipid organization and trafficking within the dynamic lung environment.     

Production in Escherichia coli of a recombinant C-terminal truncated precursor of surfactant protein B (rproSP-BΔc). Structure and interaction with lipid interfaces

SP-B, a protein absolutely required to maintain the lungs open after birth, is synthesized in the pneumocytes as a precursor containing C-terminal and N-terminal domains flanking the mature sequence. These flanking-domains are cleaved to produce mature SP-B, coupled with its assembly into pulmonary surfactant lipid-protein complexes. In the present work we have optimized over-expression in Escherichia coli and purification of rproSP-B_ΔC, a recombinant form of human proSP-B lacking the C-terminal flanking peptide, which is still competent to restore SP-B function in vivo. rProSP-B_ΔC has been solubilized, purified and refolded from bacterial inclusion bodies in amounts of about 4 mg per L of culture. Electrophoretic mobility, immunoreactivity, N-terminal sequencing and peptide fingerprinting all confirmed that the purified protein had the expected mass and sequence. Once refolded, the protein was soluble in aqueous buffers. Circular dichroism and fluorescence emission spectra of bacterial rproSP-B_ΔC indicated that the protein is properly folded, showing around 32% α-helix and a mainly hydrophobic environment of its tryptophan residues. Presence of zwitterionic or anionic phospholipids vesicles caused changes in the fluorescence emission properties of rproSP-B_ΔC that were indicative of lipid-protein interaction. The association of this SP-B precursor with membranes suggests an intrinsic amphipathic character of the protein, which spontaneously adsorbs at air-liquid interfaces either in the absence or in the presence of phospholipids. The analysis of the structure and properties of recombinant proSP-B_ΔC in surfactant-relevant environments will open new perspectives on the investigation of the mechanisms of lipid and protein assembly in surfactant complexes.     

Calcium Ions as “Miscibility Switch”: Colocalization of Surfactant Protein B with Anionic Lipids under Absolute Calcium Free Conditions

One of the main determinants of lung surfactant function is the complex interplay between its protein and lipid components. The lipid specificity of surfactant protein B (SP-B), however, and the protein's ability to selectively squeeze out lipids, has remained contradictory. In this work we present, for the first time to our knowledge, by means of time-of-flight secondary ion mass spectrometry chemical imaging, a direct evidence for colocalization of SP-B as well as its model peptide KL₄ with negatively charged dipalmitoylphosphatidylglycerol under absolute calcium free conditions. Our results prove that protein/lipid localization depends on the miscibility of all surfactant components, which itself is influenced by subphase ionic conditions. In contrast to our earlier studies reporting SP-B/KL₄ colocalization with zwitterionic dipalmitoylphosphatidylcholine, in the presence of even the smallest traces of calcium, we finally evidence an apparent reversal of protein/lipid mixing behavior upon calcium removal with ethylene diamine tetraacetic acid. In addition, scanning force microscopy measurements reveal that by depleting the subphase from calcium ions the protrusion formation ability of SP-B or KL₄ is not hampered. However, in the case of KL₄, distinct differences in protrusion morphology and height are visible. Our results support the idea that calcium ions act as a “miscibility switch” in surfactant model systems and probably are one of the major factors steering lipid/protein mixing behavior as well as influencing the protein's protrusion formation ability.     

Residue specific partitioning of KL4 into phospholipid bilayers

KL₄, which has demonstrated success in the treatment of respiratory distress, is a synthetic helical, amphipathic peptide mimetic of lung surfactant protein B. The unusual periodicity of charged residues within KL₄ and its relatively high hydrophobicity distinguish it from canonical amphipathic helical peptides. Here we utilized site specific spin labeling of both lipids and the peptide coupled with EPR spectroscopy to discern the effects of KL₄ on lipid dynamics, the residue specific dynamics of hydrophobic regions within KL₄, and the partitioning depths of specific KL₄ residues into the DPPC/POPG and POPC/POPG lipid bilayers under physiologically relevant conditions. KL₄ induces alterations in acyl chain dynamics in a lipid-dependent manner, with the peptide partitioning more deeply into DPPC-rich bilayers. Combined with an earlier NMR study of changes in lipid dynamics on addition of KL₄ (V.C. Antharam et al., 2009), we are able to distinguish how KL₄ affects both collective bilayer motions and intramolecular acyl chain dynamics in a lipid-dependent manner. EPR power saturation results for spin labeled lipids demonstrate that KL₄ also alters the accessibility profiles of paramagnetic colliders in a lipid-dependent manner. Measurements of dynamics and depth parameters for individual spin-labeled residues within KL₄ are consistent with a model where the peptide partitions deeply into the lipid bilayers but lies parallel to the bilayer interface in both lipid environments; the depth of partitioning is dependent on the degree of lipid acyl chain saturation within the bilayer.     

Interactions of the C-terminus of lung surfactant protein B with lipid bilayers are modulated by acyl chain saturation

Lung surfactant protein B (SP-B) is critical to minimizing surface tension in the alveoli. The C-terminus of SP-B, residues 59-80, has much of the surface activity of the full protein and serves as a template for the development of synthetic surfactant replacements. The molecular mechanisms responsible for its ability to restore lung compliance were investigated with circular dichroism, differential scanning calorimetry, and ³¹P and ²H solid-state NMR spectroscopy. SP-B_59-80 forms an amphipathic helix which alters lipid organization and acyl chain dynamics in fluid lamellar phase 4:1 DPPC:POPG and 3:1 POPC:POPG MLVs. At higher levels of SP-B_59-80 in the POPC:POPG lipid system a transition to a nonlamellar phase is observed while DPPC:POPG mixtures remain in a lamellar phase. Deuterium NMR shows an increase in acyl chain order in DPPC:POPG MLVs on addition of SP-B_59-80; in POPC:POPG MLVs, acyl chain order parameters decrease. Our results indicate SP-B_59-80 penetrates deeply into DPPC:POPG bilayers and binds more peripherally to POPC:POPG bilayers. Similar behavior has been observed for KL₄, a peptide mimetic of SP-B which was originally designed using SP-B_59-80 as a template and has been clinically demonstrated to be successful in treating respiratory distress syndrome. The ability of these helical peptides to differentially partition into lipid lamellae based on their degree of monounsaturation and subsequent changes in lipid dynamics suggest a mechanism for lipid organization and trafficking within the dynamic lung environment.     

Interaction of the C-Terminal Peptide of Pulmonary Surfactant Protein B (SP-B) with a Bicellar Lipid Mixture Containing Anionic Lipid

The hydrophobic lung surfactant SP-B is essential for respiration. SP-B promotes spreading and adsorption of surfactant at the alveolar air-water interface and may facilitate connections between the surface layer and underlying lamellar reservoirs of surfactant material. SP-B_63–78 is a cationic and amphipathic helical peptide containing the C-terminal helix of SP-B. ²H NMR has been used to examine the effect of SP-B_63–78 on the phase behavior and dynamics of bicellar lipid dispersions containing the longer chain phospholipids DMPC-d ₅₄ and DMPG and the shorter chain lipid DHPC mixed with a 3∶1∶1 molar ratio. Below the gel-to-liquid crystal phase transition temperature of the longer chain components, bicellar mixtures form small, rapidly reorienting disk-like particles with shorter chain lipid components predominantly found around the highly curved particle edges. With increasing temperature, the particles coalesce into larger magnetically-oriented structures and then into more extended lamellar phases. The susceptibility of bicellar particles to coalescence and large scale reorganization makes them an interesting platform in which to study peptide-induced interactions between lipid assemblies. SP-B_63–78 is found to lower the temperature at which the orientable phase transforms to the more extended lamellar phase. The peptide also changes the spectrum of motions contributing to quadrupole echo decay in the lamellar phase. The way in which the peptide alters interactions between bilayered micelle structures may provide some insight into some aspects of the role of full-length SP-B in maintaining a functional surfactant layer in lungs.     

The role of charged amphipathic helices in the structure and function of surfactant protein B.

Surfactant protein B (SP-B) is essential for normal lung surfactant function. Theoretical models predict that the disulfide cross-linked, N- and C-terminal domains of SP-B fold as charged amphipathic helices, and suggest that these adjacent helices participate in critical surfactant activities. This hypothesis is tested using a disulfide-linked construct (Mini-B) based on the primary sequences of the N- and C-terminal domains. Consistent with theoretical predictions of the full-length protein, both isotope-enhanced Fourier transform infrared (FTIR) spectroscopy and molecular modeling confirm the presence of charged amphipathic alpha-helices in Mini-B. Similar to that observed with native SP-B, Mini-B in model surfactant lipid mixtures exhibits marked in vitro activity, with spread films showing near-zero minimum surface tensions during cycling using captive bubble surfactometry. In vivo, Mini-B shows oxygenation and dynamic compliance that compare favorably with that of full-length SP-B. Mini-B variants (i.e. reduced disulfides or cationic residues replaced by uncharged residues) or Mini-B fragments (i.e. unlinked N- and C-terminal domains) produced greatly attenuated in vivo and in vitro surfactant properties. Hence, the combination of structure and charge for the amphipathic alpha-helical N- and C-terminal domains are key to SP-B function.     

Production in Escherichia coli of a recombinant C-terminal truncated precursor of surfactant protein B (rproSP-B Delta C). Structure and interaction with lipid interfaces

SP-B, a protein absolutely required to maintain the lungs open after birth, is synthesized in the pneumocytes as a precursor containing C-terminal and N-terminal domains flanking the mature sequence. These flanking-domains are cleaved to produce mature SP-B, coupled with its assembly into pulmonary surfactant lipid-protein complexes. In the present work we have optimized over-expression in Escherichia coli and purification of rproSP-B(DeltaC), a recombinant form of human proSP-B lacking the C-terminal flanking peptide, which is still competent to restore SP-B function in vivo. rProSP-B(DeltaC) has been solubilized, purified and refolded from bacterial inclusion bodies in amounts of about 4 mg per L of culture. Electrophoretic mobility, immunoreactivity, N-terminal sequencing and peptide fingerprinting all confirmed that the purified protein had the expected mass and sequence. Once refolded, the protein was soluble in aqueous buffers. Circular dichroism and fluorescence emission spectra of bacterial rproSP-B(DeltaC) indicated that the protein is properly folded, showing around 32% alpha-helix and a mainly hydrophobic environment of its tryptophan residues. Presence of zwitterionic or anionic phospholipids vesicles caused changes in the fluorescence emission properties of rproSP-B(DeltaC) that were indicative of lipid-protein interaction. The association of this SP-B precursor with membranes suggests an intrinsic amphipathic character of the protein, which spontaneously adsorbs at air-liquid interfaces either in the absence or in the presence of phospholipids. The analysis of the structure and properties of recombinant proSP-B(DeltaC) in surfactant-relevant environments will open new perspectives on the investigation of the mechanisms of lipid and protein assembly in surfactant complexes.     

Combined effects of polymers and KL4 peptide on surface activity of pulmonary surfactant lipids

Addition of ionic and nonionic polymers can improve the function of therapeutic surfactants in vitro and in vivo, especially under conditions that tend to inhibit surfactant activity. Since surfactant proteins also act to reduce surfactant inhibition, we studied the relative effects of a synthetic peptide (that mimics some of the properties of a surfactant protein), polymers, and their combination on function of surfactant phospholipid activity in vitro. We evaluated surface activity after adding polymers—polyethylene glycol or hyaluronan—to a lipid mixture with or without the synthetic peptide, sinapultide (KL₄). Using a pulsating bubble surfactometer, we measured peptide/polymer effects separately or combined at two peptide concentrations. Phospholipid mixtures, with or without KL₄ or polymers, all demonstrated good surface activity. With serum present as an inhibiting agent, adding either concentration of KL₄ reduced inhibition. Mixtures containing the higher concentration of KL₄ required higher concentrations of serum for inhibition to occur. Adding either polymer to mixtures with KL₄ further decreased susceptibility to inhibition (required higher serum concentrations). In the presence of serum, high molecular weight hyaluronan with KL₄ at 0.4 mg/ml improved surface activity to a greater degree than 0.8 mg/ml KL₄ without polymer. If the beneficial effects of adding polymer to KL₄-lipid mixtures are also borne out in the treatment of experimental lung injury, these peptide-polymer surfactant combinations may eventually prove useful in the treatment of some forms of acute lung injury in humans.     

A membranotropic region in the C-terminal domain of Hepatitis C virus protein NS4B: Interaction with membranes

We have identified a membrane-active region in the HCV NS4B protein by studying membrane rupture induced by a NS4B-derived peptide library on model membranes. This segment corresponds to one of two previously predicted amphipathic helix and define it as a new membrane association domain. We report the binding and interaction with model membranes of a peptide patterned after this segment, peptide NS4B_H2, and show that NS4B_H2 strongly partitions into phospholipid membranes, interacts with them, and is located in a shallow position in the membrane. Furthermore, changes in the primary sequence cause the disruption of the hydrophobicity along the structure and prevent the resulting peptide from interacting with the membrane. Our results suggest that the region where the NS4B_H2 is located might have an essential role in the membrane replication and/or assembly of the viral particle through the modulation of the replication complex. Our findings therefore identify an important region in the HCV NS4B protein which might be implicated in the HCV life cycle and possibly in the formation of the membranous web.     

Orientation and depth of surfactant protein B C-terminal helix in lung surfactant bilayers

SP-B(CTERM) is a cationic amphipathic helical peptide and functional fragment composed of residues 63 to 78 of surfactant protein B (SP-B). Static oriented and magic angle spinning solid state NMR, along with molecular dynamics simulation was used to investigate its structure, orientation, and depth in lipid bilayers of several compositions, namely POPC, DPPC, DPPC/POPC/POPG, and bovine lung surfactant extract (BLES). In all lipid environments the peptide was oriented parallel to the membrane surface. While maintaining this approximately planar orientation, SP-B(CTERM) exhibited a flexible topology controlled by subtle variations in lipid composition. SP-B(CTERM)-induced lipid realignment and/or conformational changes at the level of the head group were observed using (31)P solid-state NMR spectroscopy. Measurements of the depth of SP-B(CTERM) indicated the peptide center positions ~8? more deeply than the phosphate headgroups, a topology that may allow the peptide to promote functional lipid structures without causing micellization upon compression.     

Molecular dynamics study of the lung surfactant peptide SP-B1-25 with DPPC monolayers: insights into interactions and peptide position and orientation

We have performed molecular dynamics simulations of the interactions of the peptide SP-B(1-25), which is a truncated version of the full pulmonary surfactant protein SP-B, with dipalmitoylphosphatidylcholine monolayers, which are the major lipid components of lung surfactant. Simulations of durations of 10-20 ns show that persistent hydrogen bonds form between the donor atoms of the protein and the acceptors of the lipid headgroup and that these bonds determine the position, orientation, and secondary structure of the peptide in the membrane environment. From an ensemble of initial conditions, the most probable equilibrium orientation of the alpha-helix of the peptide is predicted to be parallel to the interface, matching recent experimental results on model lipid mixtures. Simulations of a few mutated analogs of SP-B(1-25) also suggest that the charged amino acids are important in determining the position of the peptide in the interface. The first eight amino acids of the peptide, also known as the insertion sequence, are found to be essential in reducing the fluctuations and anchoring the peptide in the lipid/water interface.     

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.     

Membrane surface charge modulates lipoprotein complex forming capability of peptides derived from the C-terminal domain of apolipoprotein E

Apolipoprotein E (apoE) plays a major role in the transport and metabolism of lipid by acting as a ligand for low density lipoprotein-receptors. The amphipathic helical regions of its C-terminal domain are necessary for the lipoprotein binding and assembly of nascent lipoprotein particles. Lipoproteins in the plasma are known to possess a net negative charge, determined by both its protein and lipid components, which regulates the metabolism of lipoproteins. The role of membrane surface charge on the interaction of apoE has not been studied previously. Also the importance of individual amphipathic helical regions of its C-terminal domain in binding to negatively charged lipid membrane is not addressed. In this study we have compared the interaction of four peptide segments of apoE C-terminal domain (apoE_(202-223), apoE_(223-244), apoE_(245-266), and apoE_(268-289)) with zwitterionic and negatively charged model membranes by employing UV-visible and fluorescence spectroscopy, circular dichroism, and native PAGE analysis. Our results show that the peptide sequence 202-223, 245-266 and 268-289 of apoE has higher affinity towards negatively charged lipid membrane and are independently capable of forming lipoprotein particles of 17 ± 2 nm Stokes diameter. The results suggest that surface charge of lipoprotein regulates its metabolism possibly by modulating the recruitment of apoE on its surface.     

Membrane interaction of chrysophsin-1, a histidine-rich antimicrobial peptide from red sea bream

Chrysophsin-1 is an amphipathic alpha-helical antimicrobial peptide produced in the gill cells of red sea bream. The peptide has broad range activity against both Gram-positive and Gram-negative bacteria but is more hemolytic than other antimicrobial peptides such as magainin. Here we explore the membrane interaction of chrysophsin-1 and determine its toxicity, in vitro, for human lung fibroblasts to obtain a mechanism for its antimicrobial activity and to understand the role of the unusual C-terminal RRRH sequence. At intermediate peptide concentrations, solid-state NMR methods reveal that chrysophsin-1 is aligned parallel to the membrane surface and the lipid acyl chains in mixed model membranes are destabilized, thereby being in agreement with models where permeabilization is an effect of transient membrane disruption. The C-terminal RRRH sequence was shown to have a large effect on the insertion of the peptide into membranes with differing lipid compositions and was found to be crucial for pore formation and toxicity of the peptide to fibroblasts. The combination of biophysical data and cell-based assays suggests likely mechanisms involved in both the antibiotic and toxic activity of chrysophsins.     

Critical structure-function determinants within the N-terminal region of pulmonary surfactant protein SP-B

Surfactant protein SP-B is absolutely required for the surface activity of pulmonary surfactant and postnatal lung function. The results of a previous study indicated that the N-terminal segment of SP-B, comprising residues 1–9, is specifically required for surface activity, and suggested that prolines 2, 4, and 6 as well as tryptophan 9, may constitute essential structural motifs for protein function. In this work, we assessed the role of these two motifs in promoting the formation and maintenance of surface-active films. Three synthetic peptides were synthesized including a peptide corresponding to the N-terminal 37 amino acids of native SP-B and two variants in which prolines 2, 4, 6, or tryptophan 9 were substituted by alanines. All three synthetic peptides were surface-active, as expected from their amphipathic structure. The peptides were also able to insert into dipalmitoylphosphatidylcholine/palmitoyloleoylphosphatidylglycerol (7:3 w/w ratio) monolayers preformed at pressures >30 mN/m, indicating that they perturb and insert into membranes. Substitution of alanine for tryptophan at position 9 significantly decreased both the rate of adsorption/insertion of the peptide into the interface and reinsertion of surface-active material excluded from the film during successive compression-expansion cycles. Substitution of alanines for prolines at positions 2, 4, and 6 did not produce substantial changes in the rate of adsorption/insertion; however, reinsertion of surface-active material into the expanding interface film was not as effective as in the presence of the nativelike peptide. These results suggest that W9 is critical for optimal interface affinity, whereas prolines may promote a conformation that facilitates rapid insertion of the peptide into phospholipid monolayers compressed to the highest pressures during compression-expansion cycling.     

Membrane interactions of peptides representing the polybasic regions of three Rho GTPases are sensitive to the distribution of arginine and lysine residues

Rho GTPases are a multifunctional family of proteins that are localized at cellular membranes via an isoprenyl group covalently linked to a C-terminal cysteine. Close to this primary site of membrane anchoring there is often found an additional polybasic region (PBR), which plays a secondary role in membrane binding and targeting of the complex. Here, peptides derived from the PBRs of the Rho family proteins Rac1 (K¹⁸³KRKRK), TCL (K¹⁹⁸KKKKR) and Cdc42 (P¹⁸²KKSRR) were prepared with hexalysine (K₆) and hexaarginine (R₆) to study their interactions with multilamellar vesicles of phosphatidylglycerol (DOPG) and headgroup-deuterated dimyristoylphosphatidylcholine (DMPC-d₄) using ²H and ³¹P NMR. The membranes retained their lamellar architecture after peptide binding, but the ²H NMR line shapes for DMPC-d₄ indicated that the bound peptides altered the orientation of the choline headgroups, consistent with a change in membrane surface charge. Rac1 and TCL peptides appeared to affect the headgroup orientation similarly to K₆, although the perturbations were weaker and unlike those induced by the Cdc42 peptide and R₆. Magic-angle spinning ³¹P NMR spectra of the membranes showed significant and selective broadening of the peak for DMPC after addition of the peptides, with R₆ and the Cdc42 peptide having the greatest effect. The selective broadening may be a consequence of the lipids separating into short-lived domains enriched in peptide-bound DOPG and peptide-free DMPC. These results illustrate that a complex relationship exists between the sequence of PBRs and their behaviour at membrane surfaces, which may have implications for the cellular functions and localization of Rho GTPases.