All-atom molecular dynamics simulations using orientational constraints from anisotropic NMR samples

Orientational constraints obtained from solid state NMR experiments on anisotropic samples are used here in molecular dynamics (MD) simulations for determining the structure and dynamics of several different membrane-bound molecules. The new MD technique is based on the inclusion of orientation dependent pseudo-forces in the COSMOS-NMR force field. These forces drive molecular rotations and re-orientations in the simulation, such that the motional time-averages of the tensorial NMR properties approach the experimentally measured parameters. The orientational-constraint-driven MD simulations are universally applicable to all NMR interaction tensors, such as chemical shifts, dipolar couplings and quadrupolar interactions. The strategy does not depend on the initial choice of coordinates, and is in principle suitable for any flexible molecule. To test the method on three systems of increasing complexity, we used as constraints some deuterium quadrupolar couplings from the literature on pyrene, cholesterol and an antimicrobial peptide embedded in oriented lipid bilayers. The MD simulations were able to reproduce the NMR parameters within experimental error. The alignment of the three membrane-bound molecules and some aspects of their conformation were thus derived from the NMR data, in good agreement with previous analyses. Furthermore, the new approach yielded for the first time the distribution of segmental orientations with respect to the membrane and the order parameter tensors of all three systems

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

3D Hydrophobic Moment Vectors as a Tool to Characterize the Surface Polarity of Amphiphilic Peptides

AbstractThe interaction of membranes with peptides and proteins is largely determined by their amphiphilic character. Hydrophobic moments of helical segments are commonly derived from their two-dimensional helical wheel projections, and the same is true for β-sheets. However, to the best of our knowledge, there exists no method to describe structures in three dimensions or molecules with irregular shape. Here, we define the hydrophobic moment of a molecule as a vector in three dimensions by evaluating the surface distribution of all hydrophilic and lipophilic regions over any given shape. The electrostatic potential on the molecular surface is calculated based on the atomic point charges. The resulting hydrophobic moment vector is specific for the instantaneous conformation, and it takes into account all structural characteristics of the molecule, e.g., partial unfolding, bending, and side-chain torsion angles. Extended all-atom molecular dynamics simulations are then used to calculate the equilibrium hydrophobic moments for two antimicrobial peptides, gramicidin S and PGLa, under different conditions. We show that their effective hydrophobic moment vectors reflect the distribution of polar and nonpolar patches on the molecular surface and the calculated electrostatic surface potential. A comparison of simulations in solution and in lipid membranes shows how the peptides undergo internal conformational rearrangement upon binding to the bilayer surface. A good correlation with solid-state NMR data indicates that the hydrophobic moment vector can be used to predict the membrane binding geometry of peptides. This method is available as a web application on http://www.ibg.kit.edu/HM/

Synergistic Insertion of Antimicrobial Magainin-Family Peptides in Membranes Depends on the Lipid Spontaneous Curvature

AbstractPGLa and magainin 2 (MAG2) are amphiphilic antimicrobial peptides from frog skin with known synergistic activity. The orientation of the two helices in membranes was studied using solid-state 15N-NMR, for each peptide alone and for a 1:1 mixture of the peptides, in a range of different lipid systems. Two types of orientational behavior emerged. 1), In lipids with negative spontaneous curvature, both peptides remain flat on the membrane surface, when assessed both alone and in a 1:1 mixture. 2), In lipids with positive spontaneous curvature, PGLa alone assumes a tilted orientation but inserts into the bilayer in a transmembrane alignment in the presence of MAG2, whereas MAG2 stays on the surface or gets only slightly tilted, when observed both alone and in the presence of PGLa. The behavior of PGLa alone is identical to that of another antimicrobial peptide, MSI-103, in the same lipid systems, indicating that the curvature-dependent helix orientation is a general feature of membrane-bound peptides and also influences their synergistic intermolecular interactions

Bilayer thickness determines the alignment of model polyproline helices in lipid membranes

Our understanding of protein folds relies fundamentally on the set of secondary structures found in the proteomes. Yet, there also exist intriguing structures and motifs that are underrepresented in natural biopolymeric systems. One example is the polyproline II helix, which is usually considered to have a polar character and therefore does not form membrane spanning sections of membrane proteins. In our work, we have introduced specially designed polyproline II helices into the hydrophobic membrane milieu and used 19F NMR to monitor the helix alignment in oriented lipid bilayers. Our results show that these artificial hydrophobic peptides can adopt several different alignment states. If the helix is shorter than the thickness of the hydrophobic core of the membrane, it is submerged into the bilayer with its long axis parallel to the membrane plane. The polyproline helix adopts a transmembrane alignment when its length exceeds the bilayer thickness. If the peptide length roughly matches the lipid thickness, a coexistence of both states is observed. We thus show that the lipid thickness plays a determining role in the occurrence of a transmembrane polyproline II helix. We also found that the adaptation of polyproline II helices to hydrophobic mismatch is in some notable aspects different from α-helices. Finally, our results prove that the polyproline II helix is a competent structure for the construction of transmembrane peptide segments, despite the fact that no such motif has ever been reported in natural systems.DFG, 207100805, FOR 1805: Einfluss der Ribosomendynamik auf Regulation der Geschwindigkeit und Genauigkeit der TranslationTU Berlin, Open-Access-Mittel - 201

Therapeutic Potential of Gramicidin S in the Treatment of Root Canal Infections

An intrinsic clindamycin-resistant Enterococcus faecalis, the most common single species present in teeth after failed root canal therapy, often possesses acquired tetracycline resistance. In these cases, root canal infections are commonly treated with Ledermix® paste, which contains demeclocycline, or the new alternative endodontic paste Odontopaste, which contains clindamycin; however, these treatments are often ineffective. We studied the killing activity of the cyclic antimicrobial peptide gramicidin S (GS) against planktonic and biofilm cells of tetracycline-resistant clinical isolates of E. faecalis. The high therapeutic potential of GS for the topical treatment of problematic teeth is based on the rapid bactericidal effect toward the biofilm-forming, tetracycline-resistant E. faecalis. GS reduces the cell number of planktonic cells within 20–40 min at a concentration of 40–80 μg/mL. It kills the cells of pre-grown biofilms at concentrations of 100–200 μg/mL, such that no re-growth is possible. The translocation of the peptide into the cell interior and its complexation with intracellular nucleotides, including the alarmon ppGpp, can explain its anti-biofilm effect. The successful treatment of persistently infected root canals of two volunteers confirms the high effectiveness of GS. The broad GS activity towards resistant, biofilm-forming E. faecalis suggests its applications for approval in root canal medication

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