Peptide Surfactants Research Articles

Effects of fluid-fluid interfacial elasticity on droplet formation in microfluidic devices

Biomolecules adsorb at fluid–fluid interfaces and can form a cohesive interfacial network imparting distinctive local interfacial mechanics. This modification of interfacial behavior is empirically known to significantly affect the stability and flow behavior of foams and emulsions. Droplet formation in the presence of interfacial networks is investigated in a flow-focusing microfluidic device using designed peptide surfactants, which allow decoupled control of interfacial rheology and interfacial tension. The influence of interfacial elasticity on droplet breakup, satellite droplet formation and droplet size are reported. The presence of high interfacial elasticity strongly affects the mechanism of droplet breakup by delaying neck thinning and altering interfacial shape at the point of droplet detachment, resulting in the suppression of satellite droplet formation and a decrease in droplet size. We report a correlation between dimensionless droplet size and a new dimensionless grouping which combines flow-rate ratio with the ratio of interfacial tension and interfacial elasticity. © 2010 American Institute of Chemical Engineers AIChE J, 2011

Effect of Surfactant Peptides on Electronic Properties of Single-Walled Carbon Nanotubes

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

A human surfactant peptide-elastase inhibitor construct as a treatment for emphysema

Two million Americans suffer from pulmonary emphysema, costing $2.5 billion/year and contributing to 100,000 deaths/year. Emphysema is thought to result from an imbalance between elastase and endogenous inhibitors of elastase, leading to tissue destruction and a loss of alveoli. Decades of research have still not resulted in an effective treatment other than stopping cigarette smoking, a highly addictive behavior. On the basis of our previous work, we hypothesize that small molecule inhibitors of human neutrophil elastase are ineffective because of rapid clearance from the lungs. To develop a long-acting elastase inhibitor with a lung pharmacodynamic profile that has minimal immunogenicity, we covalently linked an elastase inhibitor, similar to a trifluoro inhibitor that was used in clinical trials, to a 25-amino-acid fragment of human surfactant peptide B. We used this construct to prevent human neutrophil elastase-induced emphysema in a rodent model. The elastase inhibitor alone, although in a 70-fold molar excess to elastase in a mixture with <0.6% residual elastase activity, provided no protection from elastase-induced emphysema. Covalently combining an endogenous peptide from the target organ with a synthetic small molecule inhibitor is a unique way of endowing an active compound with the pharmacodynamic profile needed to create in vivo efficacy.

Designer Amphiphilic Short Peptides Enhance Thermal Stability of Isolated Photosystem-I

Stability of membrane protein is crucial during protein purification and crystallization as well as in the fabrication of protein-based devices. Several recent studies have examined how various surfactants can stabilize membrane proteins out of their native membrane environment. However, there is still no single surfactant that can be universally employed for all membrane proteins. Because of the lack of knowledge on the interaction between surfactants and membrane proteins, the choice of a surfactant for a specific membrane protein remains purely empirical. Here we report that a group of short amphiphilic peptides improve the thermal stability of the multi-domain protein complex photosystem-I (PS-I) in aqueous solution and that the peptide surfactants have obvious advantages over other commonly used alkyl chain based surfactants. Of all the short peptides studied, Ac-I5K2-CONH2 (I5K2) showed the best stabilizing effect by enhancing the melting temperature of PS-I from 48.0°C to 53.0°C at concentration of 0.65 mM and extending the half life of isolated PS-I significantly. AFM experiments showed that PS-I/I5K2/Triton X-100 formed large and stable vesicles and thus provide interfacial environment mimicking that of native membranes, which may partly explain why I5K2 enhanced the thermal stability of PS-I. Hydrophobic and hydrophilic group length of IxKy had an important influence on the stabilization of PS-I. Our results showed that longer hydrophobic group was more effective in stabilizing PS-I. These simple short peptides therefore exhibit significant potential for applications in membrane protein studies.

Antibacterial Activities of Short Designer Peptides: a Link between Propensity for Nanostructuring and Capacity for Membrane Destabilization

Amphiphilic peptides A(3)K, A(6)K, and A(9)K displayed an increasing propensity for nanoaggregation with increasing the size of hydrophobic alanine moiety, and the size and shape of the aggregates showed a steady transition from loose peptide stacks formed by A(3)K, long nanofibers by A(6)K, to short and narrow nanorods by A(9)K. This size and shape transition was broadly consistent with the trend predicted from interfacial packing and curvature change if these peptide surfactants were treated as conventional surfactants. The antibacterial capacity, defined by the killing of percentage of bacteria in a given time and peptide concentration, showed a strong correlation to peptide hydrophobicity, evident from both microscopic and fluorescence imaging studies. For A(9)K, the power for membrane permeation and bacterial clustering intensified with peptide concentration and incubation time. These results thus depict a positive correlation between the propensity for self-assembly of the peptides, their membrane penetration power, and bactericidal capacity. Although the exposure of A(9)K to a preformed DPPC membrane bilayer showed little structural disturbance, the same treatment to the preformed DPPG membrane bilayer led to substantial disruption of model membrane structure, a trend entirely consistent with the high selectivity observed from membrane hemolytic studies.

Interfacial Dynamic Adsorption and Structure of Molecular Layers of Peptide Surfactants

Short peptide surfactants have recently emerged as a new class of amphiphiles, with tremendous potential in improving surface biocompatibility and mediating interfacial DNA immobilization. To establish their basic interfacial adsorption properties, cationic peptide surfactants V(m)K(n) have been studied by combining the measurements of spectroscopic ellipsometry (SE), neutron reflection (NR) and atomic force microscopy (AFM). Our results showed that changes in peptide structure, concentration, solution pH and ionic strength all affected their interfacial assembly. Increases in m and decreases in n reduced the critical aggregation concentration (CAC), but increased the amount of adsorption, showing the strong influence of the amphiphilic balance between hydrophilic and hydrophobic moieties. While the surface adsorbed amount increased with time and peptide concentration, an increase in ionic strength decreased peptide adsorption due to surface charge neutralization. Changes in solution pH did not affect the equilibrium surface adsorbed amount on the weakly negative SiO(2) surface, but did alter the adsorption dynamics. Neutron reflection revealed that V(6)K readily formed a bilayer structure of 35 A thickness at the interface, with the main part of the V(6) fragments being packed back-to-back to form a 15 A hydrophobic core and the two outer K regions being incorporated with a minor amount of V fragments forming the headgroup layers of 9 A each. AFM imaging revealed a sheet-like membrane structure incorporating defects of holes but the thicknesses probed by AFM were consistent with neutron reflection. It was demonstrated that the V(6)K peptide bilayer was effective for immobilization of DNA. The amount of DNA immobilized followed approximate 1:1 charge neutralization between the outer leaf peptide sublayer and the negatively charged DNA.

Partitioning, dynamics, and orientation of lung surfactant peptide KL 4 in phospholipid bilayers

Lung surfactant protein B (SP-B) is a lipophilic protein critical to lung function at ambient pressure. KL 4 is a 21-residue peptide which has successfully replaced SP-B in clinical trials of synthetic lung surfactants. CD and FTIR measurements indicate KL 4 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 KL 4 in phospholipid bilayers, we introduced CD 3-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 KL 4 in DPPC/POPG and POPC/POPG phospholipid bilayers, as inferred from the leucine side chain dynamics, is consistent with monomeric KL 4 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 KL 4 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 KL 4. The unusual secondary structure of KL 4 and its ability to differentially partition into lipid lamellae containing varying levels of saturation suggest a mechanism for its role in restoring lung compliance.

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.

Tuneable Control of Interfacial Rheology and Emulsion Coalescence

Breaking point: Switchable peptide surfactants are used to demonstrate that the extent of cross-linking in an interfacial surfactant layer can control the rate of emulsion coalescence. Pictured is the rupture of an aqueous thin film where the peptide layer lacks sufficient strength to prevent hole formation, but nonetheless dramatically slows the rate of hole expansion.

Cover Picture: Tuneable Control of Interfacial Rheology and Emulsion Coalescence (ChemPhysChem 5/2009)

AbstractThe cover picture illustrates a microfluidic dodecane‐in‐water emulsion stabilized by the designed peptide surfactant AM1, which forms a cohesive interfacial layer through reversible interaction with ZnII. Deformed coagulated droplets are stabilized against coalescence in the presence of an elastic interfacial layer, but coalesce rapidly following ZnII chelation with EDTA. A. P. J. Middelberg et al show on page 778 that coalescence rate can be controlled by altering interfacial rheology and hence the dynamics of thin film rupture.magnified image

Streptomyces morphogenetics: dissecting differentiation in a filamentous bacterium

During the life cycle of the filamentous bacteria Streptomyces, morphological differentiation is closely integrated with fundamental growth and cell-cycle processes, as well as with truly complex multicellular behaviour that involves hormone-like extracellular signalling and coordination with an extraordinarily diverse secondary metabolism. Not only are the bacterial cytoskeleton and the machineries for cell-wall assembly, cell division and chromosome segregation reorganized during sporulation, but the developmental programme of these fascinating organisms also has many unusual elements, including the formation of a sporulating aerial mycelium and the production of a surfactant peptide and a hydrophobic sheath that allow cells to escape from the surface tension of the growth medium.

Hydrophobic-Region-Induced Transitions in Self-Assembled Peptide Nanostructures

Peptide amphiphiles readily self-assemble into a variety of nanostructures, but how molecular architectures affect the size and shape of the nanoaggregates formed is not well understood. From a combined TEM and AFM study of a series of cationic peptide surfactants AmK (m = 3, 6, and 9), we show that structural transitions (sheets, fibers/ worm-like micelles, and short rods) can be induced by increasing the length of the hydrophobic peptide region. The trend can be interpreted using the molecular packing theory developed to describe surfactant structural transitions, but the entropic gain, decreased CAC, and increased electrostatic interaction associated with increasing the peptide hydrophobic chain need to be taken into account appropriately. Our analysis indicates that the trend in structural transitions observed from AmK peptide surfactants is opposite to that obtained from conventional monovalent ionic surfactants. The outcome reflects the dominant role of hydrophobic interaction between the side chains opposed by backbone hydrogen bonding and electrostatic repulsion between lysine side chains.

Designer Peptide Surfactants Stabilize Functional Photosystem-I Membrane Complex in Aqueous Solution for Extended Time

Detailed structural analyses of membrane proteins as well as their uses in advanced nanobiotechnological applications require extended stabilization of the functional protein conformation. Here we report that a new class of designer surfactant like peptides can significantly increase the activity and stabilize the functional form of the multidomain protein complex Photosystem-I (PS-I) in solution better than other commonly used chemical detergents. We carried out a systematic analysis using a series of such peptides to identify the chemical and structural features that enhance the photochemical activity of PS-I. We observed that peptide surfactant amphiphilicity is necessary but not sufficient to stabilize PS-I in its functional form. A number of factors are essential for designing the optimal peptide including amino acid sequence, N-terminal acetylation and C-terminal amidation. Furthermore, we showed that the polarity and number of charges on the hydrophilic head are important as well as hydrophobicity and size of the amino acid side groups in the hydrophobic tail play an important role. The best performing peptides for the stabilization of functional PS-I are, in order of effectiveness, ac-I(6)K(2)-CONH(2), ac-A(6)K-CONH(2), ac-V(6)K(2)-CONH(2), and ac-V(6)R(2)-CONH(2). These simple and inexpensive peptide surfactants will likely make significant contributions to stabilize the functional form of diverse and currently elusive membrane proteins and their complexes with important applications.

Self-Assembly of Nanodonut Structure from a Cone-Shaped Designer Lipid-like Peptide Surfactant

We report here the donut-shaped nanostructure formation from the self-assembly of a designer lipid-like amphiphilic cone-shaped peptide. The critical aggregation concentration was measured using dynamic light scattering in water and phosphate-buffered saline. The dynamic self-assembly of the peptide was also studied using atomic force microscopy. We have studied numerous peptides over 17 years, and this is the first time that we have ever observed the nanodonut structure from cone-shaped peptides. We propose a plausible self-assembling pathway of the nanodonut structure that was self-assembled through the fusion or elongation of spherical micelles. Furthermore, the bending of the nanostructure gives rise to the nanodonut structures as a result of the tension originating from the interaction of the cone-shaped peptide side chains. Our observations may be useful for further fine tuning the geometry and shape of a new class of designer peptides and their self-assembled supramolecular materials for diverse uses.

Synergistic Effect and Hierarchical Nanostructure Formation in Mixing Two Designer Lipid‐Like Peptide Surfactants Ac‐A6D‐OH and Ac‐A6K‐NH2

We here report the nanostructures from combinational self-assembly of two designer lipid-like peptides Ac-A6D-OH and Ac-A6K-NH2 using dynamic light scattering (DLS) and atomic force microscopy (AFM). The synergistic phenomenon is observed by measuring the critical aggregation concentrations (CACs) of these two mixed peptides, in different molar ratios by DLS. The nanoropes were observed in AFM images at a molar ratio of Ac-A6D-OH/Ac-A6K-NH2 = 1:1, and the thin film formation with aligned nanoropes is shown at a molar ratio of 2:1. The well aligned nanoropes at the molar ratio of Ac-A6D-OH/Ac-A6K-NH2 = 2:1 indicated the competition factor between the electrostatic repulsion according to DLVO theory and the hydrophobic interaction arising from the long side chains on lysine residues. This study will further our understanding for designing new nanomaterials based on designer lipid-like peptide surfactants.

The Surfactant Peptide KL4 Sequence Is Inserted with a Transmembrane Orientation into the Endoplasmic Reticulum Membrane

Surfactant protein B (SP-B) is an essential component of pulmonary surfactant. Synthetic surfactant peptide KL4, a peptide based on a C-terminal amphipathic helical region of human SP-B, efficiently mimics some functional properties of SP-B and is included in therapeutic surfactant preparations used in trials to treat respiratory distress syndrome. The membrane orientation of this peptide is controversial. We used an in vitro transcription-translation system to study the insertion of hydrophobic sequences into microsomal membranes, and showed that the KL4 sequence integrates efficiently with a transmembrane orientation despite the presence of intermittent lysines throughout the sequence. In contrast, the precise sequence of the C-terminal SP-B amphipathic region failed to integrate, indicating a nontransmembrane orientation. Differences in the membrane insertion between KL4 and the SP-B-inspiring sequence match predictions from calculated free energies of insertion of the two sequences into membranes.

Microbial bio‐production of a recombinant stimuli‐responsive biosurfactant

Biosurfactants have been the subject of recent interest as sustainable alternatives to petroleum-derived compounds in areas ranging from soil remediation to personal and health care. The production of naturally occurring biosurfactants depends on the presence of complex feed sources during microbial growth and requires multicomponent enzymes for synthesis within the cells. Conversely, designed peptide surfactants can be produced recombinantly in microbial systems, enabling the generation of improved variants by simple genetic manipulation. However, inefficient downstream processing is still an obstacle for the biological production of small peptides. We present the production of the peptide biosurfactant GAM1 in recombinant E. coli. Expression was performed in fusion to maltose binding protein using chemically defined minimal medium, followed by a single-step affinity capture and enzymatic cleavage using tobacco etch virus protease. Different approaches to the isolation of peptide after cleavage were investigated, with special emphasis on rapid and simple procedures. Solvent-, acid-, and heat-mediated precipitation of impurities were successfully applied as alternatives to post-cleavage chromatographic peptide purification, and gave peptide purities exceeding 90%. Acid precipitation was the method of choice, due to its simplicity and the high purification factor and recovery rate achieved here. The functionality of the bio-produced peptide was tested to ensure that the resulting peptide biosurfactant was both surface active and able to be triggered to switch between foam-stabilizing and foam-destabilizing states.

Peptides As Functional Surfactants

Peptides offer interesting alternatives to conventional surfactants in applications where renewability, biocompatibility, or added functionality may be desired. This review offers a brief overview of different classes of surface-active peptides and lipopeptides, covering molecules obtained from natural sources as well as those obtained by design. Bacterial lipopeptides are cyclic molecules containing a single fatty acyl moiety, which can exhibit ultralow interfacial tension as well as antimicrobial activities. Bacterial lipopeptides have been proposed for industrial applications such as bioremediation and oil recovery, but they suffer the dual disadvantages of being difficult to bioproduce at low cost and not being easily genetically engineered. A class of synthetic molecules related to bacterial lipopeptides are the peptide amphiphiles, in which a peptide headgroup is combined with a peptide or nonpeptide hydrophobic tail. Self-assembly of peptide amphiphiles has largely been studied in bulk solution rather than at interfaces, meaning that very little information is available on the interfacial properties of these designer molecules. A larger body of information is available for protein hydrolysates, products of the partial breakdown of low-cost proteins (usually food proteins) into a complex mixture of small peptides. Partial hydrolysis can improve the functional properties of many proteins, but the outcomes are difficult to predict or control, and useful functional properties may be associated with only a few minor components in the digest mix. Finally, designed peptide surfactants (Pepfactants), recently reported by the authors, are facially amphipathic molecules that self-assemble at fluid interfaces to give cohesive films stabilizing foams and emulsions. A change in the bulk solution conditions can switch off the interfacial film, leading to rapid foam or emulsion collapse. Pepfactants can be genetically engineered and bioproduced using standard methods, which represents an advantage over bacterial lipopeptides. While peptides have not been widely used in surfactant applications so far, recent developments may facilitate the incorporation of these interesting molecules into industrial and consumer products in the near future.

Modulation of lipidic nanostructures by designer short peptide surfactants

Modulation of lipidic nanostructures by designer short peptide surfactants

Mixed System of Eudragit S-100 with a Designed Amphipathic Peptide: Control of Interfacial Elasticity by Solution Composition

We report an interfacially active system based on an informational peptide surfactant mixed with an oppositely charged polyelectrolyte. The 21-residue cationic peptide, AM1, has previously been shown to respond reversibly to pH and metal ions at fluid interfaces, forming elastic films that can be rapidly switched to collapse foams or emulsions on demand. Here we report the reversible association of AM1 with the methacrylate-based anionic polymer Eudragit S-100. The strength of the association, in bulk aqueous solution, is modulated by added metal ions and by ionic strength. Addition of zinc ions to the peptide-polymer system promotes complex formation and phase separation, while addition of a chelating agent reverses the association. The addition of salt weakens peptide-polymer interactions in the presence or absence of zinc. At the air-water interface, Eudragit S-100 forms an elastic mixed film with AM1 in the absence of metal, under conditions where the peptide alone does not show interfacial elasticity. When zinc is present, the elasticity of the mixed film is increased, but the rate of interfacial adsorption slows due to formation of peptide-polymer complexes in bulk solution. An understanding of these interactions can be used to identify favorable foam-forming conditions in the mixed system.