Antibiotic Potential and Biophysical Characterization of Amphipathic β-Stranded [XZ]n Peptides With Alternating Cationic and Hydrophobic Residues

Cationic membrane-active peptides are considered to be promising candidates for antibiotic treatment. Many natural and artificial sequences show an antimicrobial activity when they are able to take on an amphipathic fold upon membrane binding, which in turn perturbs the integrity of the lipid bilayer. Most known structures are α-helices and β-hairpins, but also cyclic knots and other irregular conformations are known. Linear β-stranded antimicrobial peptides are not so common in nature, but numerous model sequences have been designed. Interestingly, many of them tend to be highly membranolytic, but also have a significant tendency to self-assemble into β-sheets by hydrogen-bonding. In this minireview we examine the literature on such amphipathic peptides consisting of simple repetitive sequences of alternating cationic and hydrophobic residues, and discuss their advantages and disadvantages. Their interactions with lipids have been characterized with a number of biophysical techniques—especially circular dichroism, fluorescence, and infrared—in order to determine their secondary structure, membrane binding, aggregation tendency, and ability to permeabilize vesicles. Their activities against bacteria, biofilms, erythrocytes, and human cells have also been studied using biological assays. In line with the main scope of this Special Issue, we attempt to correlate the biophysical results with the biological data, and in particular we discuss which properties (length, charge, aggregation tendency, etc.) of these simple model peptides are most relevant for their biological function. The overview presented here offers ideas for future experiments, and also suggests a few design rules for promising β-stranded peptides to develop efficient antimicrobial agents

DNA-Directed Assembly of a Cell-Responsive Biohybrid Interface for Cargo Release

The development of a DNA-based cell-responsive biohybrid interface that can be used for spatially confined release of molecular cargo is reported. To this end, tailored DNA–protein conjugates are designed as gatekeepers that can be specifically cleaved by matrix metalloproteases (MMPs), which are secreted by many cancer cells. These gatekeepers can be installed by DNA hybridization on the surface of mesoporous silica nanoparticles (MSNs). The MSNs display another orthogonal DNA oligonucleotide that can be exploited for site-selective immobilization on solid glass surfaces to yield micropatterned substrates for cell adhesion. Using the human fibrosarcoma cell line HT1080 that secretes MMPs, it is demonstrated that the biohybrid surface is specifically modified by the cells to release both MSN-bound gatekeeper proteins and the encapsulated cargo peptide KLA. In view of the enormously high modularity of the system presented here, this approach promising for applications in drug delivery, tissue engineering, or other areas of nanobiotechnology is considered

Orthogonal protein decoration of DNA nanostructures based on SpyCatcher–SpyTag interaction

We present an efficient and readily applicable strategy for the covalent ligation of proteins to DNA origami by using the SpyCatcher–SpyTag (SC–ST) connector system. This approach showed orthogonality with other covalent connectors and has been used exemplarily for the immobilization and study of stereoselective ketoreductases to gain insight into the spatial arrangement of enzymes on DNA nanostructures

Membrane interactions of latarcins: Antimicrobial peptides from spider venom

A group of seven peptides from spider venom with diverse sequences constitute the latarcin family. They have been described as membrane-active antibiotics, but their lipid interactions have not yet been addressed. Using circular dichroism and solid-state 15N-NMR, we systematically characterized and compared the conformation and helix alignment of all seven peptides in their membrane-bound state. These structural results could be correlated with activity assays (antimicrobial, hemolysis, fluorescence vesicle leakage). Functional synergy was not observed amongst any of the latarcins. In the presence of lipids, all peptides fold into amphiphilic α-helices as expected, the helices being either surface-bound or tilted in the bilayer. The most tilted peptide, Ltc2a, possesses a novel kind of amphiphilic profile with a coiled-coil-like hydrophobic strip and is the most aggressive of all. It indiscriminately permeabilizes natural membranes (antimicrobial, hemolysis) as well as artificial lipid bilayers through the segregation of anionic lipids and possibly enhanced motional averaging. Ltc1, Ltc3a, Ltc4a, and Ltc5a are efficient and selective in killing bacteria but without causing significant bilayer disturbance. They act rather slowly or may even translocate towards intracellular targets, suggesting more subtle lipid interactions. Ltc6a and Ltc7, finally, do not show much antimicrobial action but can nonetheless perturb model bilayers

Extending the hydrophobic mismatch concept to amphiphilic membranolytic peptides

A series of nine amphiphilic, pore-forming α-helical KIA peptides (KIAGKIA repeats) with lengths between 14 and 28 residues were studied by solidstate 15N NMR to determine their alignment in oriented lipid bilayers. In a 2:1 mixture of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) with its corresponding 1- myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (lyso-MPC), which has a highly positive spontaneous curvature, the helix tilt angle was found to vary steadily with peptide length. The shortest peptide was aligned transmembrane and upright, while the longer ones successively became tilted away from the membrane normal. This behavior is in agreement with the hydrophobic matching concept, conceived so far only for hydrophobic helices. In 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, with a negative spontaneous curvature, all KIA peptides remained flat on the bilayer surface, while the cylindrical DMPC lipids permitted a slight tilt. Peptide insertion thus depends critically on the intrinsic lipid curvature, and helix orientation is then fine-tuned by membrane thickness. A refined toroidal pore model is proposed

Probing and Manipulating the Lateral Pressure Profile in Lipid Bilayers Using Membrane-Active Peptides—A Solid-State 19F NMR Study

The lateral pressure profile constitutes an important physical property of lipid bilayers, influencing the binding, insertion, and function of membrane-active peptides, such as antimicrobial peptides. In this study, we demonstrate that the lateral pressure profile can be manipulated using the peptides residing in different regions of the bilayer. A $^{19}$F-labeled analogue of the amphiphilic peptide PGLa was used to probe the lateral pressure at different depths in the membrane. To evaluate the lateral pressure profile, we measured the orientation of this helical peptide with respect to the membrane using solid-state $^{19}$F-NMR, which is indicative of its degree of insertion into the bilayer. Using this experimental approach, we observed that the depth of insertion of the probe peptide changed in the presence of additional peptides and, furthermore, correlated with their location in the membrane. In this way, we obtained a tool to manipulate, as well as to probe, the lateral pressure profile in membranes

Helix fraying and lipid-dependent structure of a short amphipathic membrane-bound peptide revealed by solid-state NMR

The amphipathic a-helical peptide KIA14 [(KIAGKIA)(2)-NH2] was studied in membranes using circular dichroism and solid-state NMR spectroscopy to obtain global as well as local structural information. By analyzing H-2 NMR data from 10 analogues of KIA14 that were selectively labeled with Ala-d(3), those positions that are properly folded into a helix could be determined within the membrane-bound peptide. The N-terminus was found to be unraveled, whereas positions 4-14 formed an ideal helix all the way to the C-terminus. The helicity did not change when Gly residues were replaced by Ala-d3 but was reduced when Ile was replaced, indicating that large hydrophobic residues are required for membrane binding and helix formation. The reduced helicity was strongly correlated with a decrease in peptide-induced leakage from lipid vesicles. The orientation of the short KIA14 peptide was assessed in several lipid systems and compared with that of the longer KIA21 sequence [(KIAGKIA)(3)-NH2]. In 1,2-dioleoylsn-glycero-3-phosphatidylcholine, both peptides are aligned flat on the membrane surface, whereas in 1,2-dimyristoyl-sn-glycero3-phosphatidylcholine (DMPC)/1-myristoy1-2-hydroxy-sn-glycero-3-phosphatidylcholine (lyso-MPC) both are inserted into the membrane in an upright orientation. These two types of lipid systems had been selected for their strongly negative and positive spontaneous curvature, respectively. We propose that in these cases, the peptide orientation is largely determined by the lipid properties. On the other hand, in plain DMPC and 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine, which have only a slight positive curvature, a marked difference in orientation is evident: the short KIA14 lies almost flat on the membrane surface, whereas the longer KIA21 is more tilted. We thus propose that out of the lipid systems tested here, DMPC (with hardly any curvature) is the least biased,lipid system in which peptide orientation and realignment can be studied, allowing to compare and discriminate the intrinsic effects of the properties of the peptides as such