Christopher Aisenbrey

Background/Objectives: Antimicrobial peptides are promising agents for combating resistant infections. They exhibit bactericidal activity against a wide range of microbes, primarily by disrupting the permeability of the bacterial membrane and ultimately causing cell death. Effective bacterial killing requires a high number of membrane-bound peptide molecules. Therefore, it is conceivable that peptide accumulation on the membrane could also interfere with essential cellular processes by altering bilayer dynamics, a hypothesis referred to as the “sand-in-a-gearbox” model. Methods: We systematically investigated how membrane dynamics is affected by a set of well-characterized yet highly diverse peptides: the natural AMP magainin 2, the toxin melittin, the synthetic peptides LAH4 and Killer-FLIP, and small membrane-active peptidomimetics with bactericidal activity. These effects were examined using fluorescence spectroscopy techniques, by measuring anisotropy, generalized polarization, and excimer formation of specific probes inserted at different depths within the lipid bilayer. Results: The activity of all compounds extends beyond membrane permeabilization, and the perturbation of membrane dynamics, often associated with the so-called “sand-in-a-gearbox” mechanism, is a common feature among all systems analyzed. The membrane-active compounds induced a stiffening of the phospholipid bilayer by reducing lipid lateral mobility and decreasing water penetration, at least on the nanosecond timescale accessible to fluorescence measurements. Conclusions: The concentration range in which this behavior occurred was the same for all compounds studied. This threshold is, generally, higher than that required for membrane permeabilization and reflects near-complete coverage of the bilayer surface.

Antimicrobial peptides are promising agents for combating resistant infections. They exhibit bactericidal activity against a wide range of microbes, primarily by disrupting the permeability of the bacterial membrane and ultimately causing cell death. Effective bacterial killing requires a high number of membrane-bound peptide molecules. Therefore, it is conceivable that peptide accumulation on the membrane could also interfere with essential cellular processes by altering bilayer dynamics, a hypothesis referred to as the “sand in the gearbox” model. In this work, we systematically investigated how membrane dynamics is affected by a set of well-characterized yet highly diverse peptides: the natural AMP magainin 2, the toxin melittin, the synthetic peptides LAH4 and Killer-FLIP, and small membrane-active peptidomimetics with bactericidal activity. These effects were examined using fluorescence spectroscopy techniques, by measuring anisotropy, generalized polarization, and excimer formation of specific probes inserted at different depths within the lipid bilayer. Our results show that the activity of all compounds extends beyond membrane permeabilization, and that perturbation of membrane dynamics is a common feature among all systems analyzed. The membrane-active compounds induced a stiffening of the phospholipid bilayer by reducing lipid lateral mobility and decreasing water penetration, at least on the nanosecond timescale accessible to fluorescence measurements. Interestingly, when accounting for differences in the resulting membrane surface area coverage, the concentration range in which this behavior occurred was the same for all compounds studied. This threshold is, generally, higher than that required for membrane permeabilization and reflects near-complete coverage of the bilayer surface.

Background/Objectives: This study investigates the structural and biophysical properties of the wild-type antimicrobial peptide LyeTx I, isolated from the venom of the spider Lycosa erythrognatha, and its analog LyeTx I-b, designed to enhance antibacterial activity, selectivity, and membrane interactions by the acetylation and increased amphipathicty. Methods: To understand the mechanisms behind these enhanced properties, comparative analyses of the structural, topological, biophysical, and thermodynamic aspects of the interactions between each peptide and phospholipid bilayers were evaluated. Both peptides were isotopically labeled with ²H₃-Ala and ¹⁵N-Leu to facilitate structural studies via NMR spectroscopy. Results: Circular dichroism and solid-state NMR analyses revealed that, while both peptides adopt α-helical conformations in membrane mimetic environments, LyeTx I-b exhibits a more amphipathic and extended helical structure, which correlates with its enhanced membrane interaction. The thermodynamic properties of the peptide–membrane interactions were quantitatively evaluated in the presence of phospholipid bilayers using ITC and DSC, highlighting a greater propensity of LyeTx I-b to disrupt lipid vesicles. Calcein release studies reveal that both peptides cause vesicle disruption, although DLS measurements and TEM imaging indicate distinct effects on phospholipid vesicle organization. While LyeTx I-b permeabilizes anionic membrane retaining the vesicle integrity, LyeTx I promotes significant vesicle agglutination. Furthermore, DSC and calcein release assays indicate that LyeTx I-b exhibits significantly lower cytotoxicity toward eukaryotic membranes compared to LyeTx I, suggesting greater selectivity for bacterial membranes. Conclusions: Our findings provide insights into the structural and functional modifications that enhance the antimicrobial and therapeutic potential of LyeTx I-b, offering valuable guidance for the design of novel peptides targeting resistant bacterial infections and cancer.

The widely used Laurdan probe has two conformers, resulting in different optical properties when embedded in a lipid bilayer membrane, as demonstrated by our previous simulations. Up to now, the two conformers’ optical responses have, however, not been investigated when the temperature and the phase of the membrane change. Since Laurdan is known to be both a molecular rotor and a solvatochromic probe, it is subject to a profound interaction with both neighboring lipids and water molecules. In the current study, molecular dynamics simulations and hybrid Quantum Mechanics/Molecular Mechanics calculations are performed for a DPPC membrane at eight temperatures between 270K and 320K, while the position, orientation, fluorescence lifetime and fluorescence anisotropy of the embedded probes are monitored. The importance of both conformers is proven through a stringent comparison with experiments, which corroborates the theoretical findings. It is seen that for Conf-I, the excited state lifetime is longer than the relaxation of the environment, while for Conf-II, the surroundings are not yet adapted when the probe returns to the ground state. Throughout the temperature range, the lifetime and anisotropy decay curves can be used to identify the different membrane phases. The current work might, therefore, be of importance for biomedical studies on diseases, which are associated with cell membrane transformations.

Background: SAAP-148 is an antimicrobial peptide derived from LL-37. It exhibits excellent activity against drug-resistant bacteria and biofilms while resisting degradation in physiological conditions. Despite its optimal pharmacological properties, its mechanism of action at the molecular level has not been explored. Methods: The structural properties of SAAP-148 and its interaction with phospholipid membranes mimicking mammalian and bacterial cells were studied using liquid and solid-state NMR spectroscopy as well as molecular dynamics simulations. Results: SAAP-148 is partially structured in solution and stabilizes its helical conformation when interacting with DPC micelles. The orientation of the helix within the micelles was defined by paramagnetic relaxation enhancements and found similar to that obtained using solid-state NMR, where the tilt and pitch angles were determined based on ¹⁵N chemical shift in oriented models of bacterial membranes (POPE/POPG). Molecular dynamic simulations revealed that SAAP-148 approaches the bacterial membrane by forming salt bridges between lysine and arginine residues and lipid phosphate groups while interacting minimally with mammalian models containing POPC and cholesterol. Conclusions: SAAP-148 stabilizes its helical fold onto bacterial-like membranes, placing its helix axis almost perpendicular to the surface normal, thus probably acting by a carpet-like mechanism on the bacterial membrane rather than forming well-defined pores.

The accumulation of aggregated protein is a typical hallmark of many human neurodegenerative disorders, including polyglutamine-related diseases such as chorea Huntington. Misfolding of the amyloidogenic proteins gives rise to self-assembled complexes and fibres. The huntingtin protein is characterised by a segment of consecutive glutamines which, when exceeding ~ 37 residues, results in the occurrence of the disease. Furthermore, it has also been demonstrated that the 17-residue amino-terminal domain of the protein (htt17), located upstream of this polyglutamine tract, strongly correlates with aggregate formation and pathology. Here, we demonstrate that membrane interactions strongly accelerate the oligomerisation and β-amyloid fibril formation of htt17-polyglutamine segments. By using a combination of biophysical approaches, the kinetics of fibre formation is investigated and found to be strongly dependent on the presence of lipids, the length of the polyQ expansion, and the polypeptide-to-lipid ratio. Finally, the implications for therapeutic approaches are discussed.

Magainin 2 and PGLa are cationic, amphipathic antimicrobial peptides which when added as equimolar mixture exhibit a pronounced synergism in both their antibacterial and pore-forming activities. Here we show for the first time that the peptides assemble into defined supramolecular structures along the membrane interface. The resulting mesophases are quantitatively described by state-of-the art fluorescence self-quenching and correlation spectroscopies. Notably, the synergistic behavior of magainin 2 and PGLa correlates with the formation of hetero-domains and an order-of-magnitude increased membrane affinity of both peptides. Enhanced membrane association of the peptide mixture is only observed in the presence of phophatidylethanolamines but not of phosphatidylcholines, lipids that dominate bacterial and eukaryotic membranes, respectively. Thereby the increased membrane-affinity of the peptide mixtures not only explains their synergistic antimicrobial activity, but at the same time provides a new concept to increase the therapeutic window of combinatorial drugs.

The major histocompatibility complex class II (MHC II) membrane proteins are key players in the adaptive immune response. An aberrant function of these molecules is associated with a large number of autoimmune diseases such as diabetes type I and chronic inflammatory diseases. The MHC class II is assembled from DQ alpha 1 and DQ beta 1 which come together as a heterodimer through GXXXG-mediated protein–protein interactions and a highly specific protein-sphingomyelin-C18 interaction motif located on DQA1. This association can have important consequences in regulating the function of these membrane proteins. Here, we investigated the structure and topology of the DQA1 and DQB1 transmembrane helical domains by CD-, oriented ²H and ¹⁵N solid-state NMR spectroscopies. The spectra at peptide-to-lipid ratios of 0.5 to 2 mol% are indicative of a topological equilibrium involving a helix crossing the membrane with a tilt angle of about 20° and another transmembrane topology with around 30° tilt. The latter is probably representing a dimer. Furthermore, at the lowest peptide-to-lipid ratio, a third polypeptide population becomes obvious. Interestingly, the DQB1 and to a lesser extent the DQA1 transmembrane helical domains exhibit a strong fatty acyl chain disordering effect on the inner segments of the ²H-labelled palmitoyl chain of POPC bilayers. This phosphatidylcholine disordering requires the presence of sphingomyelin-C18 suggesting that the ensemble of transmembrane polypeptide and sphingolipid exerts positive curvature strain.

When polypeptides bind to the membrane surface, they become confined to a restricted quasi-two-dimensional space where peptide–peptide interactions become highly relevant, and the concept of a crowded medium is appropriate. Within this crowded environment interesting effects like clustering, separation of phases, cooperative alignment, and common movements occur. Here we investigated such effects by measuring distances between fluorophore-labeled peptides in the range ≤1 nm by fluorescence self-quenching. For helical peptides with dimensions of approximately 1 × 3 nm such a small “ruler” is sensitive to the packing of the labeled peptides and thereby to their molecular arrangement. A novel approach to characterize peptide–peptide interactions within membranes is presented using the designer peptide LAH4. This sequence changes membrane topology in a controlled manner being transmembrane at neutral conditions but oriented parallel to the surface at low pH. Experimental measurements of the fluorescence self-quenching of close-by chromophores and the changes that occur upon dilution with unlabeled peptides are used to analyze the peptide distribution within the membrane surface. The data show a strong effect of electrostatic interactions and under some experimental conditions clustering of the peptides. Furthermore, the results suggest that at pH 4 the peptides arrange along the membrane surface in an ordered mesophase-like arrangement.