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Scientific Reports Volume 6, Article Number: 21123 (2016) Cite this article

Membrane active peptides are of great significance for the development of drug delivery vehicles and therapies for the treatment of multi-drug resistant infections. Lack of specificity may be harmful, and finding ways to regulate the specificity and activity of membrane active peptides is essential to improve their therapeutic effects and minimize harmful side effects. We describe a de novo designed membrane-active peptide that partitions into the lipid membrane only when it is specifically and covalently anchored to the membrane, thereby forming pores. Dimerization with complementary peptides effectively inhibited the formation of pores. This effect can be regulated by matrix metalloproteinase MMP-7 which inhibits the proteolytic digestion of peptides, which is an enzyme that is upregulated in many malignant tumors. Therefore, the system provides a precise and specific way to regulate the permeability of lipid membranes, and provides a new strategy for the development of indirect enzyme controlled release based on the identified membrane active peptides and liposome cargo.

A large number of natural and designed peptides can reshape and disrupt the structure and permeability of cell membranes by triggering membrane rupture 1, 2, fusion 3, 4 or translocation 5, 6. These so-called membrane active peptides have great structural, functional and compositional diversity, but are often amphipathic and rich in positively charged amino acids. They are also easy to accumulate at the lipid membrane interface 7, 8, leading to membrane separation or cell internalization. Membrane active peptides are usually unstructured in solution, but they adopt a well-defined secondary structure when combined with lipid membranes9. An important class of membrane active peptides are antimicrobial peptides (AMP). They are part of the host’s first line of defense against multiple biological infections and show broad-spectrum antibiotic activity10. AMPs have been studied for centuries, but the occurrence of multi-drug resistance of pathogens to traditional antibiotics (including carbapenem-resistant Enterobacteriaceae 11, 12) has led to a huge demand for new antibiotics, which has renewed people’s interest in AMPs. Interests 2, 13, 14. AMP can prove to have significant effects on Gram-positive and Gram-negative bacteria, as well as fungi, enveloped viruses, parasites and cancer cells, and is therefore a potentially attractive candidate for the treatment of antibiotic-resistant bacterial infections15.

Membrane active peptides can interact with lipid membranes in a variety of ways, such as by forming transmembrane pores (barrel pores or annular pores) or by surface-related carpet mechanisms1,2,8 causing membrane disturbances. However, they usually lack specific high-affinity interactions with specific membrane components 9 and AMP, and therefore show the smallest inhibitory concentration in the micromolar range1. The initial peptide membrane binding is usually a rate-limiting process, but it can be accelerated by peptide lipidation. Lipidation increases their affinity for lipid membranes, which may improve their therapeutic effect. Modification of short peptides with lipids has converted other non-membrane active peptides into AMPs, showing antifungal and antiviral properties17,18. A significant improvement in the activity of the lipidated glycopeptide antibiotic vancomycin against resistant strains has also been reported19. However, lipidation of AMP can significantly reduce its water solubility and increase toxicity15. Therefore, a more suitable strategy to increase the affinity and selectivity of the AMP membrane is to trigger the specific and high-affinity binding of the peptide to the substances already present in the lipid membrane. This is further motivated by observations that, in addition to their bactericidal properties, AMPs can also prove to have an adverse toxic effect on healthy non-pathogenic cells and exhibit undesirable and significant hemolytic activity15. Therefore, finding ways to regulate and increase the specificity and activity of natural AMP and designed membrane active peptides is essential to avoid potential toxicity and improve its therapeutic effect. In addition, the designed membrane-active peptides with target specificity and adjustable activity are also important for liposome-mediated drug delivery, drug release, and gene transfection20. Since membrane-active peptides can regulate the integrity of lipid membranes, they may also be used to regulate the release of liposome-encapsulated drugs. The possibility of specifically regulating and triggering the release of liposome-encapsulated drugs will enable better control of the release, thereby reducing the required therapeutic dose.

Here, we describe a membrane-active helix-loop-helix peptide (JR2KC) designed de novo. When specifically and covalently anchored to the head group functionalized lipid present in the membrane, the peptide will be divided into zwitterions Lipid film. Anchoring and subsequent membrane distribution trigger the structural transition of the peptide from random coiling to α-helical conformation and pore formation, as shown in Figure 1. The degree of pore formation can be achieved by changing the number of anchors or by introducing a complementary helix-loop-helix peptide (JR2E), which is intended to heterodimerize with JR2KC and fold into a four-helix bundle 21. In addition, we show that the inhibitory activity of JR2E can be controlled enzymatically by the matrix metalloproteinase MMP-7. The up-regulation of MMP-7 is related to the progression of a series of malignant tumors, and is believed to be related to cancer metastasis and various inflammatory processes22. JR2E contains two specific recognition sites for MMP-7, which are located at 11-Ala-|-Leu-12 and 26-Ala-| Gln-27 and JR2E. The proteolytic cleavage of JR2E leads to truncated peptide 23, which cannot be combined with JR2KC. Heterodimerization. Therefore, MMP-7 reactivated the membrane permeabilization of JR2KC. Here, we therefore demonstrated a new recognition-based strategy for specifically regulating the permeability of lipid membranes using membrane-active peptides designed de novo. In addition to demonstrating possible ways to obtain a more specific designer AMP, the system can also achieve enzyme-mediated liposome release. The latter indicates a possible strategy for designing peptides to increase the efficacy of liposome-encapsulated anticancer drugs by increasing the release rate near the tumor.

POPC liposomes (top) with different amounts (1-10 mol%) of maleimide head-functionalized lipids (MPB-PE) were prepared.

The MPB-PE lipid anchors the redesigned helix-loop-helix polypeptide JR2KC to the membrane, triggering pore formation (left). The formation of pores is inhibited by adding JR2E, a charge-complementary polypeptide that heterodimerizes with JR2KC and folds into a four-helix bundle (right). Not drawn to scale.

JR2KC and JR2E were originally designed to adopt a helix-loop-helix motif, and heterodimerize into a four-helix bundle in a solution of neutral pH. JR2KC has an amphiphilic residue distribution, with a hydrophobic surface and a highly charged polar surface rich in lysine (Supplementary Figure 1). Therefore, the characteristics of JR2KC are reminiscent of many natural AMPs, which usually have both cationic and amphiphilic properties14, 24, 25, and 26. Exposing carboxyfluorescein (CF)-loaded liposomes prepared from zwitterion POPC to JR2KC (4 μM) showed that the peptide has a limited effect on membrane integrity, resulting in only a small release of CF (Figure 2). In order to promote specific peptide membrane binding maleimide head group functionalized lipids (MPB-PE) are included in the membrane at different concentrations. The maleimide moiety selectively reacts with the thiol group in the cysteine ​​residue in the peptide loop region through Michael addition, thereby covalently anchoring the peptide to the lipid in the membrane (Supplementary Figure 2) .

As indicated by the arrows, after 30 minutes of incubation with JR2KC (blue) using 1, 3, 5, and 10 mol% MPB-PE, carboxyfluorescein (CF) is released from the liposomes. The increase in MPB-PE concentration resulted in a wider release of JR2KC.

Liposomes containing 0 mol% MPB-PE (orange) have limited release, similar to JR2K (green), oxidized JR2KC (grey), and hybrid peptides (purple) that use liposomes containing 5 mol% MPB-PE. Note that the orange and purple curves overlap. All data points are average values ​​of n = 3 and SD ≤ 2%.

Compared with liposomes without maleimide, liposomes prepared with as little as 1 mole percent (mol%) of MPB-PE have observed a significant increase in CF release (Figure 2). After 30 minutes of incubation, less than 10% CF release was observed from POPC liposomes without maleimide anchor (0 mol% MPB-PE) in the presence of 4 μM JR2KC. In the presence of MPB-PE, CF release is significant and highly dependent on the amount of MPB-PE and the concentration of JR2KC. Increasing the peptide concentration from 0-4 μM resulted in faster release kinetics and broader release (Figure 2 and Supplementary Figure 3). Increasing the amount of MPB-PE in the membrane can increase the release rate and significantly reduce the peptide concentration required to trigger the release of CF. In the absence of peptides, the CF release of all liposome compositions is negligible (Supplementary Figure 4). When valine is used to replace the cysteine ​​residue in JR2KC, it has no effect on membrane integrity and prevents the peptide from covalently binding to the maleimide part of MPB-PE (Figure 2). When the cysteine ​​in JR2KC is oxidized to form two disulfide-linked monomers, CF release is also eliminated. Compared with natural AMP, JR2KC-mediated membrane permeabilization is therefore very specific and effective, releasing about 90% of peptides within 30 minutes, and only about 100 nM of peptides. Therefore, these experiments show that by selectively and site-specifically anchoring JR2KC to the membrane, a substantial and adjustable increase in membrane permeability can be obtained.

Use circular dichroism (CD) spectroscopy to study the effect of membrane interactions on the secondary structure of peptides. In the absence and presence of maleimide (0% MPB-PE) liposomes, JR2KC is randomly coiled at neutral pH (Figure 3A). In the presence of liposomes containing 5 mol% MPB-PE, the CD spectrum of JR2KC shows significant helicity, with a minimum at 208 and 222 nm, and an average residual at 222 nm ([θ]222) The basic ellipticity is about -15000° cm2 dmol−1. The initial transition from random coil to α-helix is ​​very fast within a few minutes, but the helicity continues to increase within about 10 minutes after the peptide is added (Figure 3B). Therefore, the folding kinetics is comparable to the CF release rate (Supplementary Figure 3). The folding of the peptide indicates that membrane anchoring induces the partitioning of JR2KC into the hydrophobic core of the membrane, which facilitates the induction of secondary structure through partition-folding coupling, as observed in many AMPs. When the primary sequence of JR2KC is disturbed, membrane permeabilization is eliminated, leaving only the position of cysteine ​​residues to form non-amphiphilic peptides (Figure 2 and Supplementary Figure 1). Compared with JR2KC, the peptide does not fold in the presence of MPB-PE-containing liposomes (Figure 3), which clearly shows that, except for the need to anchor the peptide to the membrane. Dynamic light scattering (DLS) experiments showed that the liposome size (hydrodynamic radius) of liposomes with and without MPB-PE only slightly changed in the presence of JR2KC (Supplementary Figure 5). Therefore, the binding of peptides to liposomes does not trigger the formation of micelles, nor does it trigger the aggregation of liposomes.

CD spectra of JR2KC (10 μM in PBS pH 7.4) in the absence (orange, dotted line) and the presence of 0 mol% (orange) and 5 mol% (blue) POPC liposomes of MPB-PE.

Scrambled peptides (10 μM in PBS pH 7.4) show no ordered secondary structure in the absence (purple, dashed line) or the presence of POPC (purple) with 5 mol% MPB-PE. Inset: [θ]222 and [θ]208 of JR2KC (10 μM in PBS pH 7.4) in the presence of POPC liposomes with 0 mol% (orange) and 5 mol% (blue) MPB-PE The rate of change over time.

In order to further characterize the interaction between JR2KC and lipid bilayers, we used a quartz crystal microbalance (QCM-D) with dissipative function to examine the effect of peptide adsorption on the uniform planar support lipid bilayer (SLB). By correlating mass changes measured under different overtones, corresponding to different penetration depths, QCM-D can provide a mechanical assessment of peptide-mediated membrane destruction27,28,29. SLB is formed by the collapse of spontaneous vesicles on the SiO2 coated sensor crystal, as shown by a frequency shift (Δf) of ~25 Hz and a dissipation drift (ΔD) close to zero. Figure 4A shows the QCM-D trajectories of Δf and ΔD at the 3rd to 11th overtones during the injection of 4 μM JR2KC on a POPC double layer with 5 mol% MPB-PE. An initial slight increase in Δf and ΔD (position (4) in Figure 4A) is observed, followed by a significant and rapid decrease in Δf and an increase in ΔD, until a steady state is reached (Δf = -38 Hz and ΔD = 9.5 × 10-6 is the third overtone). The large frequency shift indicates that the covalent attachment of JR2KC to MPB-PE leads to further peptide recruitment to the membrane. The initial small increase in Δf and ΔD at all overtones indicates a certain loss of lipids caused by peptide binding and possible pore formation, which makes the membrane more hydrated and less rigid. The small loss of lipids may be the result of increased membrane tension during peptide distribution, which cannot be compensated by a flat-supported lipid bilayer compared to liposomes that can swell to accommodate additional materials. This event is followed by a large decrease in Δf and an increase in ΔD at all overtones, indicating that more peptides are attached, which increases the total mass of the surface while reducing the stiffness of the membrane. It is worth noting that within the first 60 seconds of injection of JR2KC, the slopes of Δf and ΔD for different overtones are almost the same (Figure 4A and Supplementary Figure 6), which indicates that the initial changes in quality and viscoelasticity throughout the membrane, that is, peptides may be inserted Into the membrane, a transmembrane pore 28 is formed. JR2KC concentrations from 1 μM to 8 μM gave similar responses, Δf (~38 Hz) and ΔD (~10-5) (Figure 5). However, the binding kinetics was significantly slower at lower peptide concentrations (Figure 4B), which may indicate that the membrane concentration-dependent synergistic effect involves peptide binding and insertion. The resulting overtone-dependent Δf and ΔD shifts indicate that most of the peptides are trapped on the membrane surface, resulting in uneven changes in the mass and viscoelasticity of the entire bilayer (Figure 5). Importantly, membrane removal or destruction did not occur even at high peptide concentrations (8 μM), which is consistent with the DLS results, indicating that a detergent-like carpet mechanism is unlikely to serve as a mode of action. In addition, when the POPC bilayer without the maleimide moiety was exposed to 8 μM JR2KC (Supplementary Fig. 7a), no membrane binding was observed, confirming that specific membrane anchoring is required for JR2KC to distribute and fold. When the membrane-bound JR2KC was exposed to the charge-complementing peptide JR2E, no further mass increase was observed (Supplementary Figure 7b), indicating that JR2KC could not undergo dimerization, possibly due to the distribution of JR2KC into the membrane. On the other hand, when JR2KC and JR2E were allowed to heterodimerize before adding MPB-PE liposomes containing CF loading, a sharp reduction in CF release was observed (Figure 6). At a concentration of 10 μM JR2E, the membrane activity of JR2KC is more or less eliminated. Therefore, the formation of heterodimer four-helix bundles dynamically and specifically prevents the possibility of JR2KC inserting into lipid membranes. Using different JR2E concentrations, the membrane permeabilization of JR2KC can be adjusted very accurately and repeatedly (Figure 6B).

QCM-D measurement (A) anchors 4 μM JR2KC to an SLB composed of POPC and 5 mol% MPB-PE, displaying overtones 3, 5, 7, 9 and 11 (from dark to light gray). (1) PBS buffer, (2) inject POPC liposomes containing 5 mol% MPB-PE to form SLB, (3) rinse with PBS, (4) inject 4 μM JR2KC, and finally (5) rinse with PBS. (B) After injection of 1, 2, 4, and 8 μM (from light blue to dark blue) JR2KC on SLB composed of POPC and 5 mol% MPB-PE, the third overtone shows the response of Δf.

QCM-D changes in Δf and ΔD caused by JR2KC injected into SLB containing POPC at (A,B) 1 μM, (C,D) 2 μM, (E,F) 4 ​​μM and (G,H) 8 μM concentrations Contains 5 mol% MPB-PE.

(A) Schematic diagram of the inhibitory effect on JR2KC after adding the complementary peptide JR2E (top). These two peptides fold into a helix-loop-helix motif and dimerize into a four-helix bundle. Dimerization and folding effectively prevent JR2KC from forming holes. JR2E can be proteolytically degraded by MMP-7, thereby restoring the pore-forming ability of JR2KC (bottom). (B) JR2E concentration-dependently inhibits JR2KC-triggered CF leakage from POPC liposomes with 5 mol% MPB-PE and 1 μM JR2KC, n = 3. (C) JR2KC triggers the release of CF after degradation of JR2E (50 μM) with 0, 0.5, 1, 2, 5, and 10 μg/mLMMP-7 for 2 hours, normalized to the CF release of 1 μM JR2KC. After adding to the CF-loaded POPC liposomes containing 5 mol% MPB-PE, the final concentration is 1 μM JR2KC and 5 μM JR2E.

JR2E contains two MMP-7 recognition sites (11-Ala-|-Leu-12 and 26-Ala-| Gln-27), and proteolytic cleavage greatly reduces the ability of JR2E to heterodimerize with JR2KC23. The incubation of JR2E and MMP-7 resulted in the MMP-7 concentration-dependent recovery of JR2KC membrane activity. When added to liposomes containing 5 mol% MPB-PE, MMP-7 alone (1 μg/ml) caused only a slight increase in CF release (4.6 ± 0.2%) (Supplementary Figure 8). When JR2E was exposed to 10 μg/ml MMP-7 for 2 hours before mixing with JR2KC, the inhibitory capacity of the peptide was reduced by approximately 50%. Therefore, in the presence of MMP-7, the substantial increase in JR2KC membrane permeability is a direct result of the increase in the concentration of non-dimerized JR2KC caused by JR2E hydrolytic digestion, and membrane partitioning can be carried out.

The possibility of specifically binding membrane-active peptides to cell-type-dependent biomarkers on the cell surface may be a very interesting strategy to increase the selectivity of AMP and other membrane-active peptides of interest in therapeutic applications. In this work, we used the recognition of head group modification lipids to enhance and adjust the membrane destruction effect of the de novo designed AMP-like helix-loop-helix peptide (JR2KC). Our research results show that recognition-based membrane anchoring is indeed a feasible and effective strategy that can improve the specificity and membrane permeability of membrane-active peptides, which are limited by low efficacy and limited selectivity. The combination of a de novo designed membrane active peptide with a thiol-reactive maleimide head group functionalized lipid (MPB-PE) specifically significantly increases membrane permeability by forming pores. The 42-amino acid helix-loop-helix peptide has two fragments of approximately 20 residues flanking cysteine ​​residues in the loop region that reacts with MPB-PE. Both fragments meet the length requirement of the peptide to form a transmembrane pore spanning the lipid bilayer (> 20 residues). The covalent anchoring of JR2KC and MPB-PE increases the effective concentration of the peptide at the membrane-water interface and reduces its conformational flexibility, promoting distribution and pore formation. The degree and kinetics of the release can be reasonably adjusted by the maleimide surface concentration and the peptide to lipid ratio, where an increase in any of these parameters will result in faster and more extensive membrane destruction. The increase in the efficacy of membrane active peptides that depend on the surface concentration of specific recognition moieties is a very desirable feature for future therapeutic applications.

In addition, we demonstrated the possibility of adjusting the activity of this membrane-active peptide through specific and competitive folding-dependent interaction with the complementary peptide (JR2E). The two peptides JR2KC and JR2E are designed to fold into a helix-loop-helix motif and heterodimerize into a four-helix bundle. The heterodimerization event acts as a switch to inhibit the pore-forming ability of JR2KC, presumably because hydrophobic residues are hidden in the hydrophobic core of the four-helix bundle, preventing partition folding coupling. The dissociation constant of the heterodimer four-helix bundle in solution is in the micromolar range (0.02 mM), and the dimerization event shows a fast switching rate31. At a concentration of 10 μM JR2E and 1 μM JR2KC, there should be approximately 0.7 μM JR2KC available for pore formation. Therefore, the concentration required for JR2E to inhibit JR2KC is slightly lower than the concentration estimated from the dissociation constant of the free peptide. This may be the result of the pre-concentration effect of charge-charge regulation leading to the accumulation of peptides (JR2E) in the membrane boundary area. Since the formation of heterodimers is completely reversible, the inhibitory effect of JR2E can be reversed. We include two MMP-7 recognition sites in the primary sequence of JR2E, located on both sides of the loop region. Proteolytic cleavage greatly reduces the size of the peptide, making heterodimerization very disadvantageous. Therefore, the activity of JR2KC can also be regulated by the enzymatic activity of MMP-7. The signs that the pore-forming ability of the designed peptides can be activated by clinically relevant biomarkers open up new possible strategies for adjusting the specificity and efficacy of membrane active peptides in therapeutic applications, as well as designing new enzyme-modulated drug delivery systems and in vivo sensing applications .

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl) butyramide] (MPB-PE) Purchased from Avanti Polar Lipids (Alabaster, USA). Unless otherwise stated, other chemicals were purchased from Sigma Aldrich (Singapore).

Peptides JR2KC (NAADLKKAIKALKKHLKAKGPCDAAQLKKQLKQAFKAFKRAG), JR2K (same sequence as JR2KC, but Cys at position 22 is replaced by Val) and JR2E (NAADLEKAIEALEKHLEAKGPVDAAQLEAGQLEQAFEAFER in the standard chemistry 31). The crude product was purified by reverse phase HPLC on an Ace C-8 column and identified by MALDI-TOF mass spectrometry. Jumble JR2KC (AKKLLAAKAHKDKGIKPKALNCDAKFAKFKAKQQKRA) was purchased from GL Biochem (Shanghai, China) with a purity of >95%. Assuming that the water content in the lyophilized state is 25%, the peptide concentration at the time of dissolution is estimated. The oxidation of JR2KC is carried out by incubating JR2KC (2 mg/ml) in 10 mM ammonium bicarbonate buffer at pH 8.5 at room temperature for 72 hours. Ellman test confirmed complete oxidation. The oxidized peptide was diluted in MQ water, rotary evaporated and lyophilized sequentially.

Liposomes are prepared by rehydrating the film and then extruding. The stock solutions of POPC and MPB-PE in chloroform were mixed in the following molar ratios: 100:0, 99:01, 97:03, 95:05 and 90:10. A gentle stream of nitrogen was used to evaporate the solvent before drying under vacuum overnight. Vesicles were formed by adding 1.0 mL of 0.1 M phosphate buffered saline (PBS), pH 7.4 to the dried film, incubating for 20 minutes, and then vortexing for 1 minute. Subsequently, a micro extruder (Avanti Polar Lipids) was used to extrude the vesicles through a 100 nm polycarbonate membrane etched on the trajectory 21 times to form large unilamellar vesicles. For carboxyfluorescein (CF) dye encapsulation, add 1.0 mL of 50 mM CF (self-quenching concentration) in 20 mM sodium phosphate, 10 mM NaCl (pH 7.4) to the dried lipid cake, then as described above Prepare vesicles. The total lipid is 5mg/mL. A PD-25 column (GE Healthcare, Singapore) was used to remove unencapsulated CF by gel filtration and eluted with PBS buffer.

A fluorescence microplate reader (Infinite 200, Tecan, Austria; λex = 485 nm, λem = 520 nm) was used to study peptide-induced leakage of CF liposomes. Before adding 0, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 4 μM peptide, the liposomes were diluted to 0.05 mg/mL (total lipid concentration) with PBS buffer in a 96-well plate. Starting from the self-quenching concentration of CF inside the liposome, any leakage of the dye can be detected as an increase in fluorescence intensity over time. The percentage of CF released over time is calculated as 100 × (F − F0)/(FT − F0), where F0 is the initial fluorescence of CF before peptide addition, F is the fluorescence of CF at the time interval t, and FT is the difference between 1% Triton X -100 Total fluorescence after complete release of CF after 15 minutes of incubation.

Incubate JR2E (50 μM) with MMP-7 at a concentration of 0-10 μg/mL for 2 hours at 37°C. The reaction was quenched by heating to 80°C for 20 minutes. Incubate the digested JR2E and JR2KC at room temperature for 30 minutes, then add liposomes and analyze CF leakage as described above.

The CD spectrum was recorded using a Jasco J-810 spectropolarimeter (Jasco Corporation, Japan) at 25°C using a 0.01 cm optical path CD cuvette. The CD spectrum of 10 μM JR2KC dissolved in PBS buffer (pH 7.4) was recorded in the absence or presence of liposomes containing only 0.5 mg/ml POPC or POPC containing 5 mol% MPB-PE, scanning speed It is 50 nm/min, a resolution is 0.1 nm, and the bandwidth is 1 nm. After adding the peptide to the liposomes, the spectrum was continuously recorded for 25 minutes. Use PBS for baseline correction. Average the three spectra of each sample.

The QCM-D experiment was carried out on the Q-Sense E4 instrument (Q-Sense AB, Gothenburg, Sweden). All measurements are performed on silicon dioxide-coated sensor crystals. The crystals were washed with 1% w/w sodium dodecyl sulfate (SDS) solution, and rinsed thoroughly with water and ethanol. After lightly drying under a stream of nitrogen, the crystals were treated with ultraviolet ozone cleaner for 5 minutes. For supporting lipid bilayer formation, inject 1 mL of 0.1 mg/mL liposomes dissolved in PBS, pH 7.4 at a flow rate of 100 μL/min, and then rinse with buffer for at least 10 minutes. 1, 2, 4, and 8 μM JR2KC dissolved in PBS was injected at a flow rate of 100 μL/min to study membrane fixation and distribution.

The DLS experiment was carried out using the ALV/DLS/SLS-5022F system from ALV-GmbH, Langen, Germany, using a HeNe laser of 632.8 nm with an output power of 22 mW. Use the CONTIN 2DP program implemented in the ALV data analysis package to calculate the liposome size distribution. All samples are filtered through a hydrophilic PVDF filter with a pore size of 0.2 μm before measurement. The temperature is stabilized at 21.5 °C by a constant temperature bath.

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DA, CS and RS thank Linköping University, Swedish Research Council (VR), Swedish Strategic Research Foundation (SSF), Knut and Alice Wallenberg Foundation (KAW), and Nano Science and Technology Center (CeNano) for their financial support. SKL and BL Acknowledge the financial support from the Office of the Provost of NTU. In this research, CS joined the Graduate School Forum Scientium.

Bionic Sensor Science Center, School of Materials Science and Engineering, Nanyang Technological University, Research Techno Plaza, 6th floor XFrontiers block, 50 Nanyang Drive, 637553, Singapore

Seng Koon Lim & Bo Liedberg

Department of Molecular Physics, Department of Physics, Chemistry and Biology, Linköping University, Linköping, 581 83, Sweden

Camilla Sandern, Robert Salegard and Daniel Avery

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SKL and CS made the same contribution to this work. DA conceived this work and wrote this paper with the contributions of all authors. RS synthesized peptides and performed MALDI-TOF, and SKL and CS were tested on liposomes and supporting lipid bilayers. DA and BL use SKL, CS, and RS to interpret the data. All authors agree with the final version of the manuscript.

The author declares that there are no competing economic interests.

This work has been licensed under the Creative Commons Attribution 4.0 International License Agreement. The images or other third-party materials in this article are included in the Creative Commons license of the article, unless otherwise stated in the credit line; if the material is not included under the Creative Commons license, the user will need permission from the license holder to copy The material. To view a copy of this license, please visit http://creativecommons.org/licenses/by/4.0/

Lim, S., Sandén, C., Selegård, R. etc. Competitive peptide dimerization and partition folding interactions modulated by proteolytic activity modulate liposome membrane permeability. Scientific Report 6, 21123 (2016). https://doi.org/10.1038/srep21123

DOI: https://doi.org/10.1038/srep21123

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Scientific Report (Sci Rep) ISSN 2045-2322 (online)