Exploring peptide-membrane interactions with coarse-grained MD simulations

The interaction of α-helical peptides with lipid bilayers is central to our understanding of the physicochemical principles of biological membrane organization and stability. Mutations that alter the position or orientation of an α-helix within a membrane, or that change the probability that the α-helix will insert into the membrane, can alter a range of membrane protein functions. We describe a comparative coarse-grained molecular dynamics simulation methodology, based on self-assembly of a lipid bilayer in the presence of an α-helical peptide, which allows us to model membrane transmembrane helix insertion. We validate this methodology against available experimental data for synthetic model peptides (WALP23 and LS3). Simulation-based estimates of apparent free energies of insertion into a bilayer of cystic fibrosis transmembrane regulator-derived helices correlate well with published data for translocon-mediated insertion. Comparison of values of the apparent free energy of insertion from self-assembly simulations with those from coarse-grained molecular dynamics potentials of mean force for model peptides, and with translocon-mediated insertion of cystic fibrosis transmembrane regulator-derived peptides suggests a nonequilibrium model of helix insertion into bilayers.     

Specific and selective peptide-membrane interactions revealed using quartz crystal microbalance

The skin secretions of Australian tree frogs are rich in peptides with potential antimicrobial activity. They interrupt bacterial cell membranes, although precisely how and whether all peptides have the same mechanism is not known. The interactions of three of these peptides—aurein 1.2, maculatin 1.1, and caerin 1.1 with supported phospholipid bilayers—are examined here using quartz crystal microbalance and atomic force microscopy. These approaches enabled us to reveal variations in material structure and density as a function of distance from the sensor surface when comparing mass sensorgrams over a range of harmonics of the natural resonance of the sensor crystal and hence obtain for the first time to our knowledge a mechanistic assessment of membrane disruption. We found that caerin inserted into the bilayer in a transmembrane manner, regardless of concentration and phospholipid composition consistent with a pore-forming mechanism. In contrast, maculatin and aurein interacted with membranes in a concentration-dependent manner. At low concentrations (<5 μM), maculatin exhibited transmembrane incorporation whereas aurein was limited to surface association. Upon reaching a threshold value of concentration, both peptides lysed the membrane. In the case of maculatin, the lysis progressed in a slow, concentration-dependent manner, forming mixed micelles, as shown by atomic force microscopy imaging. Aurein-induced lysis proceeded to a sudden disruption, which is consistent with the “carpet” mechanism. Both maculatin and aurein exhibit specificity toward phospholipids and thus have potential as candidates as antimicrobial drugs.     

Fluid or gel phase lipid bilayers to study peptide-membrane interactions?

Since many studies on peptide-membrane interactions are carried out only with fluid phase lipid bilayers (L _α -phase, absence of cholesterol) we have investigated whether this phase is really a suitable model for biological membranes. For this purpose the action of melittin on zwitterionic and negatively charged phospholipid bilayers, in the absence and presence of 30 mol% cholesterol, was investigated by solid state ³¹P-NMR. From the NMR point of view, it appears that systems composed of a single phospholipid best mimic the sterol-containing system a few degrees below the gel-to-fluid phase transition, i. e., in the rippled phase (P _β' ). It is then proposed that a relatively rigid membrane containing local defects, rather than a L _α -bilayer, is required as an appropriate model for natural membranes when probing the action of melittin. Such requirements might be crucial when studying peptide-lipid interactions. Received: 5 February 1996 / Accepted: 1 July 1996     

Thermodynamics of drug-DNA interactions

Many anticancer, antibiotic, and antiviral drugs exert their primary biological effects by reversibly interacting with nucleic acids. Therefore, these biomolecules represent a major target in drug development strategies designed to produce next generation therapeutics for diseases such as cancer. In order to improve the clinical efficacy of existing drugs and also to design new ones it is necessary to understand the molecular basis of drug-DNA interactions in structural, thermodynamic, and kinetic detail. The past decade has witnessed an increase in the number of rigorous biophysical studies of drug-DNA systems and considerable knowledge has been gained in the energetics of these binding reactions. This is, in part, due to the increased availability of high-sensitivity calorimetric techniques, which have allowed the thermodynamics of drug-DNA interactions to be probed directly and accurately. The focus of this article is to review thermodynamic approaches to examining drug-DNA recognition. Specifically, an overview of a recently developed method of analysis that dissects the binding free energy of these reactions into five component terms is presented. The results of applying this analysis to the DNA binding interactions of both minor groove drugs and intercalators are discussed. The solvent water plays a key role in nucleic acid structure and consequently in the binding of ligands to these biomolecules. Any rational approach to DNA-targeted drug design requires an understanding of how water participates in recognition and binding events. Recent studies examining hydration changes that accompany DNA binding by intercalators will be reviewed. Finally some aspects of cooperativity in drug-DNA interactions are described and the importance of considering cooperative effects when examining these reactions is highlighted.     

Thermodynamics of lipid-peptide interactions

This review is focused on peptide molecules which exhibit a limited solubility in the aqueous phase and bind to the lipid membrane from the aqueous medium. Surface adsorption, membrane insertion, and specific binding are usually accompanied by changes in the heat content of the system and can be measured conveniently with isothermal titration calorimetry, avoiding the necessity of peptide labeling. The driving forces for peptide adsorption and binding are hydrophobicity, electrostatics, and hydrogen bonding. An exclusively hydrophobic interaction is exemplified by the immunosuppressant drug cyclosporine A. Its insertion into the membrane can be described by a simple partition equilibrium X(b)=K(0)C(eq). If peptide and membrane are both charged, electrostatic interactions are dominant leading to nonlinear binding curves. The concentration of the peptide near the membrane interface can then be much larger than its bulk concentration. Electrostatic effects must be accounted for by means of the Gouy-Chapman theory before conventional binding models can be applied. A small number of peptides and proteins bind with very high affinity to a specific lipid species only. This is illustrated for the lantibiotic cinnamycin (Ro 09-0198) which forms a 1:1 complex with phosphatidyethanolamine with a binding constant of 10(8) M(-1). Membrane adsorption and insertion can be accompanied by conformational transitions facilitated, in part, by hydrogen bonding mechanisms. The two membrane-induced conformational changes to be discussed are the random coil-to-alpha-helix transition of amphipathic peptides and the random coil-to-beta-structure transition of Alzheimer peptides.     

Roles of hydrophobicity and charge distribution of cationic antimicrobial peptides in peptide-membrane interactions

Cationic antimicrobial peptides (CAPs) occur as important innate immunity agents in many organisms, including humans, and offer a viable alternative to conventional antibiotics, as they physically disrupt the bacterial membranes, leading to membrane lysis and eventually cell death. In this work, we studied the biophysical and microbiological characteristics of designed CAPs varying in hydrophobicity levels and charge distributions by a variety of biophysical and biochemical approaches, including in-tandem atomic force microscopy, attenuated total reflection-FTIR, CD spectroscopy, and SDS-PAGE. Peptide structural properties were correlated with their membrane-disruptive abilities and antimicrobial activities. In bacterial lipid model membranes, a time-dependent increase in aggregated β-strand-type structure in CAPs with relatively high hydrophobicity (such as KKKKKKALFALWLAFLA-NH(2)) was essentially absent in CAPs with lower hydrophobicity (such as KKKKKKAAFAAWAAFAA-NH(2)). Redistribution of positive charges by placing three Lys residues at both termini while maintaining identical sequences minimized self-aggregation above the dimer level. Peptides containing four Leu residues were destructive to mammalian model membranes, whereas those with corresponding Ala residues were not. This finding was mirrored in hemolysis studies in human erythrocytes, where Ala-only peptides displayed virtually no hemolysis up to 320 μM, but the four-Leu peptides induced 40-80% hemolysis at the same concentration range. All peptides studied displayed strong antimicrobial activity against Pseudomonas aeruginosa (minimum inhibitory concentrations of 4-32 μM). The overall findings suggest optimum routes to balancing peptide hydrophobicity and charge distribution that allow efficient penetration and disruption of the bacterial membranes without damage to mammalian (host) membranes.     

Thermodynamics of sequence-specific protein-DNA interactions

The molecular recognition processes in sequence-specific protein-DNA interactions are complex. The only feature common to all sequence-specific protein-DNA structures is a large interaction interface, which displays a high degree of complementarity in terms of shape, polarity and electrostatics. Many molecular mechanisms act in concert to form the specific interface. These include conformational changes in DNA and protein, dehydration of surfaces, reorganization of ion atmospheres, and changes in dynamics. Here we review the current understanding of how different mechanisms contribute to the thermodynamics of the binding equilibrium and the stabilizing effect of the different types of noncovalent interactions found in protein-DNA complexes. The relation to the thermodynamics of small molecule-DNA binding and protein folding is also briefly discussed.     

Thermodynamics of phospholipid-sucrose interactions.

The effect of 0-1.0 M sucrose on the phase-transition properties of 1,2-dipalmitoyl-3-sn-phosphatidylcholine (1,2-DPPC) was examined by high-sensitivity differential scanning calorimetry at a scan rate of 0.1 K min-1. Increasing the concentration of sucrose caused a small, but experimentally significant, increase in the temperature (Tm) of maximal excess apparent specific heat (Cmax) and in delta T 1/2 (the transition width at 1/2 Cmax), a reduction in Cmax, and a small decrease (approximately 8-10% at 1.0 M sucrose compared with 0 M sucrose) in the calorimetric enthalpy (delta Hcal) of the gel-to-liquid crystalline transition. The calorimetric parameters of the pretransition of 1,2-DPPC were not significantly affected by sucrose in the concentration range examined, except there was a 1.0 degree C increase in the temperature (Tp) of maximal excess apparent specific heat in the presence of 1.0 M sucrose. The results are discussed in terms of the possible molecular mechanisms that could have caused the observed changes and are contrasted with the results obtained by C. -H. Chen et al. (1981, Biophys. J., 36:359-367).     

Surface plasmon resonance spectroscopy: an emerging tool for the study of peptide-membrane interactions

The interactions between peptides and membranes mediate a wide variety of biological processes, and characterization of the molecular details of these interactions is central to our understanding of cellular events such as protein trafficking, cellular signaling and ion-channel formation. A wide variety of biophysical techniques have been combined with the use of model membrane systems to study peptide-membrane interactions, and have provided important information on the relationship between membrane-active peptide structure and their biological function. However, what has generally not been reported is a detailed analysis of the affinity of peptide for different membrane systems, which has largely been due to the difficulty in obtaining this information. To address this issue, surface plasmon resonance (SPR) spectroscopy has recently been applied to the study of biomembrane-based systems using both planar mono- or bilayers or liposomes. This article provides an overview of these recent applications that demonstrate the potential of SPR to enhance our molecular understanding of membrane-mediated peptide function.     

Thermodynamics of the alpha-helix-coil transition of amphipathic peptides in a membrane environment: implications for the peptide-membrane binding equilibrium

Amphipathic alpha-helices are the membrane binding motif in many proteins. The corresponding peptides are often random coil in solution but are folded into an alpha-helix upon interaction with the membrane. The energetics of this ubiquitous folding process are still a matter of conjecture. Here, we present a new method to quantitatively analyze the thermodynamics of peptide folding at the membrane interface. We have systematically varied the helix content of a given amphipathic peptide when bound to the membrane and have correlated the thermodynamic binding parameters determined by isothermal titration calorimetry with the alpha-helix content obtained by circular dichroism spectroscopy. The peptides investigated were the antibiotic magainin 2 amide and three analogs in which two adjacent amino acid residues were substituted by their d-enantiomers. The thermodynamic parameters controlling the alpha-helix formation were found to be linearly related to the helicity of the membrane-bound peptides. Helix formation at the membrane surface is characterized by an enthalpy change of DeltaH(helix) approximately -0.7 kcal/mol per residue, an entropy change of DeltaS(helix) approximately -1.9 cal/molK residue and a free energy change of DeltaG(helix)=-0.14 kcal/mol residue. Helix formation is a strong driving force of peptide insertion into the membrane and accounts for about 50 % of the free energy of binding. An increase in temperature entails an unfolding of the membrane-bound helix. The temperature dependence can be described with the Zimm-Bragg theory and the enthalpy of unfolding agrees with that deduced from isothermal titration calorimetry.     

Thermodynamics of carbohydrate-lipid interactions.

Thermodynamic analyses of carbohydrate-lipid interactions were performed by investigating the effects of a series of carbohydrates, including monosaccharides, disaccharides, and trisaccharides, on the phase-transition properties of aqueous dispersions of 1,2-dipalmitoyl phosphatidylcholine (DPPC). The temperature of the lipid's main phase transition from the gel to liquid-crystalline phase is essentially unchanged in the presence of carbohydrate. The change in the free energy (delta G) of the transition is zero when a carbohydrate is added to aqueous dispersions of DPPC, while the enthalpy (delta H) and the entropy of the melting of DPPC are decreased. The thermodynamic information was used to examine carbohydrate-lipid interactions. Such interactions were elucidated according to our knowledge of the specific properties of carbohydrates in aqueous solutions and the previously proposed hydrophobic interaction involving hydrocarbon tails of the lipid in aqueous dispersions.     

Lipid dependence of peptide-membrane interactions: Bilayer affinity and aggregation of the peptide alamethicin

Membrane incorporation and aggregation of the peptide alamethicin have been investigated as a function of lipid type. Head group and acyl chain regions both contribute to modulate alamethicin incorporation. Specifically, the peptide prefers thin membranes and saturated chains; incorporation is reduced by the presence of cholesterol. Aggregation of the peptide in the bilayer is virtually insensitive to changes in lipid composition. These findings show some analogies to results obtained with intrinsic membrane proteins and cast doubt on the use of global membrane parameters for interpreting lipid-peptide interactions.     

The use of fluoresceinphosphatidylethanolamine (FPE) as a real-time probe for peptide-membrane interactions

The characterization of fluorescelnphosphatidylethanolamlne (FPE) as a real-time Indicator of the electrostatic nature of a membrane surface is described. The conditions appropriate for the labelling of membranes and the implementation of FPE as a tool to monitor the interactions of various peptides with model membranes are outlined. It is shown that of the membrane-active peptides studied, Naja naja kaouthla cardiotoxin and pyrularia thionin bind to certain model membranes without insertion. Whereas the leader sequence of the nuclear encoded subunit IV of mammalian cytochrome c oxidase (E.C. 1.9.3.1), known as p-25, and melittin appear to bind and then partially insert into the membrane. It seems evident also that melittin does not adopt a fully transmembrane configuration. Melittin is known to promote membrane lysis and by employing a rapid-kinetic technique it is shown that the time-course of such lysis does not appear to correlate with peptide binding, but following binding a significant proportion of melittin must become inserted into the membrane before lysis appears to commence.     

Cellular uptake of S413-PV peptide occurs upon conformational changes induced by peptide-membrane interactions

In face of accumulated reports demonstrating that uptake of some cell-penetrating peptides occurs through previously described endocytic pathways, or is a consequence of cell fixation artifacts, we conducted a systematic analysis on the mechanism responsible for the cellular uptake of the S4(13)-PV karyophilic cell-penetrating peptide. The results reviewed here show that the S4(13)-PV peptide is able to very efficiently accumulate inside live cells in a rapid, non-toxic and dose-dependent manner, through a mechanism distinct from endocytosis. Comparative analysis of peptide uptake by mutant cells lacking heparan sulfate proteoglycans demonstrates that, although not mandatory, their presence at cell surface facilitates the cellular uptake of the S4(13)-PV peptide. Furthermore, we demonstrate that upon interaction with lipid vesicles, the S4(13)-PV peptide undergoes significant conformational changes that are consistent with the formation of helical structures. Such conformational changes occur concomitantly with a penetration of the peptide into the lipid bilayer, strongly suggesting that the resulting helical structures are crucial for the non-endocytic cellular uptake of the S4(13)-PV peptide. Overall, our data support that, rather than endocytosis, the cellular uptake of the S4(13)-PV cell-penetrating peptide is a consequence of its direct translocation through cell membranes following conformational changes induced by peptide-membrane interactions.     

Atomic detail peptide-membrane interactions: molecular dynamics simulation of gramicidin S in a DMPC bilayer

Molecular dynamics simulations have been performed of the sequence-symmetric cyclic decapeptide antibiotic gramicidin S (GS), in interaction with a hydrated dimyristoylphosphatidylcholine (DMPC) bilayer, and the results compared with a "control" simulation of the system in the absence of GS. Following experimental evidence, the GS was initially set in a single antiparallel beta-sheet conformation with two Type II' beta-turns in an amphiphilic interaction with the membrane. This conformation and position remained in the 6.5 ns simulation. Main-chain dihedrals are on average approximately 26 degrees from those determined by NMR experiment on GS in dimethylsulfoxide (DMSO) solution. Sequence-symmetric main-chain and side-chain dihedral angle pairs converge to within approximately 5 degrees and approximately 10 degrees, respectively. The area per lipid, lipid tail order parameters, and quadrupole spin-lattice relaxation times of the control simulation are mostly in good agreement with corresponding experiments. The GS has little effect on the membrane dipole potential or water permeability. However, it is found to have a disordering effect (in agreement with experiment) and a fluidifying effect on lipids directly interacting with it, and an ordering effect on those not directly interacting.     

Structure investigation of amphiphilic cyclopeptides in isotropic and anisotropic environments-A model study simulating peptide-membrane interactions.

It has been proposed that the membrane allows a much more efficient binding of certain small or medium-sized amphiphilic messenger molecules to their receptor, not only by accumulation of the drug, but also by induction of orientations and conformations that are much more favorable for receptor docking than structures adopted in isotropic phases. A series of eight amphiphilic cyclic peptides containing lipophilic (L-alpha-aminodecanoic acid = Ada, L-alpha-aminohexadecanoic acid = Ahd, Nhdg = N-hexadecylglycine) and hydrophilic (Lys, Asp) amino acids were synthesized and examined by means of NMR spectroscopy and molecular dynamics (MD) simulations in isotropic (CDCl3) and membrane-mimicking anisotropic (SDS/H2O) solvents to study the influence of the environment on their individual conformations. NMR data of cyclo(-Gly1-D-Asp2-Ahd3-Ahd4-Asp5-Gly6+ ++-) (C4), cyclo(-Lys1-D-Pro2-Lys3-Ada4-Pro5-Ada6-) (C5) and cyclo(-Lys1-Pro2-Lys3-Ada4-D-Pro5-Ada6-) (C6) clearly indicate that those compounds are too rigid to perform a conformational change upon transition from an isotropic to an anisotropic environment. On the other hand, the experimental data of cyclo (-Gly1-Asp2-Ahd3-Ahd4-Asp5-Gly6-) (C1), cyclo(-Asp1-Ala2-Nhdg3-Ala4-D-Asp5-) (C7), and cyclo(-D-Asp1-Ala2-Nhdg3-Ala4-Asp5-) (C8) suggest highly flexible unstructured molecules in both environments. However, for cyclo(-Asp1-Asp2-Gly3-Ahd4-Ahd5-Gly6-) (C2) we observed a structure inducing effect of a membrane-like environment. The compound populates three different conformations in SDS/H2O, whereas in CDCI3 no preferred conformation can be detected. cyclo(-D-Asp1-Asp2-Gly3-Ahd4-Ahd5-Gly6-) (C3) clearly exhibits two different conformations with a shifted beta,beta-turn motif in CDCI3 and SDS/H2O solutions. The conformational change could be reproduced in a restraint-free MD simulation using the biphasic membrane mimetic CCl4/H2O. Our results give clear evidence that membrane interactions may not only lead to structure inductions, but can also induce major conformational changes in compounds already exhibiting a defined structure in isotropic solution.     

Thermodynamics of lipid interactions in complex bilayers

The mutual interactions between lipids in bilayers are reviewed, including mixtures of phospholipids, and mixtures of phospholipids and cholesterol (Chol). Binary mixtures and ternary mixtures are considered, with special emphasis on membranes containing Chol, an ordered phospholipid, and a disordered phospholipid. Typically the ordered phospholipid is a sphingomyelin (SM) or a long-chain saturated phosphatidylcholine (PC), both of which have high phase transitions temperatures; the disordered phospholipid is 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) or dioleoylphosphatidylcholine (DOPC). The unlike nearest-neighbor interaction free energies (ω_AB) between lipids (including Chol), obtained by an variety of unrelated methods, are typically in the range of 0-400 cal/mol in absolute value. Most are positive, meaning that the interaction is unfavorable, but some are negative, meaning it is favorable. It is of special interest that favorable interactions occur mainly between ordered phospholipids and Chol. The interpretation of domain formation in complex mixtures of Chol and phospholipids in terms of phase separation or condensed complexes is discussed in the light of the values of lipid mutual interactions.     

Thermodynamics of alpha-lactalbumin-DL-alpha-dipalmitoylphosphatidylcholine interactions and effect of the antioxidant nicotinamide on these interactions

Differential scanning calorimetry has been used to understand the thermodynamics of the interactions of dl-alpha-dipalmitoylphosphatidylcholine (DPPC) with alpha-lactalbumin and the effect of the antioxidant nicotinamide on these interactions. Nicotinamide decreases the thermal transition temperature of both the lipid and the protein at high concentrations. The thermal unfolding transitions of the protein were two state and calorimetrically reversible. There was no significant change in the shape and thermodynamic parameters accompanying the lipid endotherms, suggesting that nicotinamide did not penetrate the lipid bilayer. The thermal unfoldings of alpha-lactalbumin in the presence of DPPC as cosolute also adhered to two-state reversible mechanism. The changes in the thermodynamic parameters accompanying the thermal transitions were small, indicating no significant interaction of alpha-lactalbumin with DPPC. The changes in the thermodynamic parameters indicate that the lipid bilayer organization, as well as the partitioning of the extrinsic protein alpha-lactalbumin into the bilayer, is not affected in the entire studied concentration range of the lipid. It is observed that the presence of increasing concentration of nicotinamide (as high as 1.0 mol dm(-3)) in the lipid-protein mixture does not affect its partitioning into the lipid bilayer, although nicotinamide preferentially interacts with alpha-lactalbumin. The change in the effect of nicotinamide on lipid transition temperature in the mixture and literature report suggests that nicotinamide may be forming a hydrogen-bonded complex with the protein through its amide functionality. The surface tension data of aqueous nicotinamide in combination with the thermal denaturation results of protein in presence of nicotinamide confirmed that surface tension effect does not have any significant contribution to the effect of nicotinamide on protein.