Membrane-water Interface Research Articles

Structural Features of Membrane-bound Glucocerebrosidase and α-Synuclein Probed by Neutron Reflectometry and Fluorescence Spectroscopy

Mutations in glucocerebrosidase (GCase), the enzyme deficient in Gaucher disease, are a common genetic risk factor for the development of Parkinson disease and related disorders, implicating the role of this lysosomal hydrolase in the disease etiology. A specific physical interaction exists between the Parkinson disease-related protein α-synuclein (α-syn) and GCase both in solution and on the lipid membrane, resulting in efficient enzyme inhibition. Here, neutron reflectometry was employed as a first direct structural characterization of GCase and α-syn·GCase complex on a sparsely-tethered lipid bilayer, revealing the orientation of the membrane-bound GCase. GCase binds to and partially inserts into the bilayer with its active site most likely lying just above the membrane-water interface. The interaction was further characterized by intrinsic Trp fluorescence, circular dichroism, and surface plasmon resonance spectroscopy. Both Trp fluorescence and neutron reflectometry results suggest a rearrangement of loops surrounding the catalytic site, where they extend into the hydrocarbon chain region of the outer leaflet. Taking advantage of contrasting neutron scattering length densities, the use of deuterated α-syn versus protiated GCase showed a large change in the membrane-bound structure of α-syn in the complex. We propose a model of α-syn·GCase on the membrane, providing structural insights into inhibition of GCase by α-syn. The interaction displaces GCase away from the membrane, possibly impeding substrate access and perturbing the active site. GCase greatly alters membrane-bound α-syn, moving helical residues away from the bilayer, which could impact the degradation of α-syn in the lysosome where these two proteins interact.

Comparing Peptide-Lipid Interactions and Antimicrobial Activities of Peptides with Similar “Core” Lengths But Variable Arginine and Tryptophan Residues

Antimicrobial peptides offer a potential alternative therapy option for the increasing problem of microbial drug resistance. To address this issue, “hybrid” peptides with total lengths intermediate between the surface-acting antimicrobial lactoferrin-derived LfB6 (RRWQWR-NH2) and the inert model transmembrane α-helical peptide RWALP23 (acetyl-GRALW(LA)6LWLARA-NH2) have been designed with varying numbers of arginines, tryptophans and net positive charge, but with the same “core” length of five residues. The lipid interactions of the hybrid LfB-RWALP peptides having between 9 and 15 residues were investigated by circular dichroism, tryptophan fluorescence, and 31P and 2H solid-state NMR spectroscopy, and their antimicrobial activities assayed. Peptide . . . . . . . . . Sequence RRWWALP15 . . . . acetyl-GRRWWLALALWWRRA15-NH2 RRWWALP13 . . . . acetyl-RRWWLALALWWRR13-NH2 RWALP11 . . . . . . . acetyl-GRWLALALWRA11-NH2 RWALP9 . . . . . . . . RWLALALWR9-NH2 Our results indicate that despite having the same core length, the peptides exhibit different conformations, different interactions with lipids, and different antimicrobial activities. While the 13-mer is α-helical in neutral and anionic lipid mixtures, the conformations of the shorter 11-mer and 9-mer vary considerably depending on the lipid environment. Fluorescence spectroscopy suggests that the tryptophan residues of all peptides are located at the membrane-water interface, with slightly varying depths of insertion into the lipid bilayer. Despite small perturbations to the phospholipid head groups, solid-state 31P NMR spectra indicate that the lipids remain primarily in a bilayer phase. 2H NMR spectra reveal that the two shorter peptides aggregate, while the two longer (and more helical) peptides align in neutral and anionic lipid mixtures. Although all four peptides exhibit similar antimicrobial activities, some variation is observed against gram negative and gram positive bacterial strains.

Detection of Helix Fraying in Designed Transmembrane Alpha Helices

Transmembrane helices of integral membrane proteins often have aromatic residues flanking them. The aromatic residues favor locations within the membrane–water interface of a lipid bilayer and may serve as anchors to aid the stabilization of a tilted transmembrane orientation. To further understand the influence of Tyr, Trp or Phe residues upon the properties of helical membrane proteins (see Biochemistry 53, 3637), we have investigated the possibility of partial unwinding near the ends of selected transmembrane helices. For this purpose, we substituted positions 4 and 5 with either two Phe or two Ala residues to generate F4,5GWALP23 (acetyl-GGAF4F5(LA)6LW19LAGA-ethanolamide) or A4,5GWALP23 (acetyl-GGAA4A5(LA)6LW19LAGA-ethanolamide), respectively. By incorporating specific 2H-Ala labels at positions 3 and 21, we are able to compare the influence of interfacial Phe and Ala on the unwinding of the helix ends. Solid state NMR spectra of macroscopically aligned bilayer samples show well defined 2H-Ala quadrupolar splittings for both F4,5GWALP23 and A4,5GWALP23, suggesting that the peptide helices are well oriented in DLPC, DMPC and DOPC lipid bilayers. We are also able to estimate the helix tilt from deuterium labeling of the core alanine residues. Geometric Analysis of Labeled Alanines (GALA) then shows unwinding at the terminals for both F4,5GWALP23 and A4,5GWALP23. It is conceivable that the helix fraying may be critical for the stability of the transmembrane helix orientation in the lipid bilayer membranes.

Direct Measurement of Dipole Electric Field in Model Membranes using Vibrtaional Shifts of P-Cyanophenylalanine and Coupled with Molecular Dynamics Simulations

The composition of biological lipid bilayer membrane creates a complex structural and electrostatic environment that regulates important membrane functions. The alignment of molecular dipole moments from lipid head groups and water molecules located at membrane-water interface creates the dipole potential (Vd). The dipole field (Fd) generated from this potential is the largest and about 1-10 MV/cm in magnitude, determined by indirect measurement techniques. It is located entirely within the membrane interior, and therefore, direct measurement and manipulation of Fd is a challenge. Here, we present direct measurement of dipole electrostatic field within vesicular bilayer using vibrational Stark effect (VSE) shifts of nitrile oscillator in response to the local electrostatic field. We intercalated four different α-helix transmembrane peptides containing unnatural amino acid, p-cyanophenylalanine (p-CN-Phe) at four unique positions into unilamellar vesicles composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Molecular Dynamics simulations of the membrane-intercalated helix containing two of the nitrile probes, one near the head-group region of the lipid and one buried in the interior of the bilayer were performed to examine the structure of nitrile with respect to the membrane normal, the assumed direction of Fd, by quantifying both tilt of the helix in bilayer and conformational rotation of the p-CN-Phe side chain at steady-state. As nitrile systematically moved towards the membrane interior, the vibrational absorption energies of nitrile showed blue shift. We used the measured VSE shifts and nitrile orientations within the membrane and calculated the magnitude of Fd to be 8 −11 MV/cm, within the range reported in the literature. We increased the chemical and structural complexity of model membrane by adding cholesterol into vesicle composition at different concentrations and determined that Fd changes as function of membrane composition and temperature.

Dynamic scenario of membrane binding process of kalata b1.

Kalata B1 (kB1), a cyclotide that has been used in medical applications, displays cytotoxicity related to membrane binding and oligomerization. Our molecular dynamics simulation results demonstrate that Trp19 in loop 5 of both monomeric and tetrameric kB1 is a key residue for initial anchoring in the membrane binding process. This residue also facilitates the formation of kB1 tetramers. Additionally, we elucidate that kB1 preferentially binds to the membrane interfacial zone and is unable to penetrate into the membrane. In particular, significant roles of amino acid residues in loop 5 and loop 6 on the localization of kB1 to this membrane-water interface zone are found. This study reveals the roles of amino acid residues in the bioactivity of kB1, which is information that can be useful for designing new therapeutic cyclotides with less toxicity.

Experimental determination and computational interpretation of biophysical properties of lipid bilayers enriched by cholesteryl hemisuccinate

Cholesteryl hemisuccinate (CHS) is one of the cholesterol-mimicking detergents not observed in nature. It is, however, widely used in protein crystallography, in biochemical studies of proteins, and in pharmacology. Here, we performed an extensive experimental and theoretical study on the behavior of CHS in lipid membranes rich in unsaturated phospholipids. We found that the deprotonated form of CHS (that is the predominant form under physiological conditions) does not mimic cholesterol very well. The protonated form of CHS does better in this regard, but also its ability to mimic the physical effects of cholesterol on lipid membranes is limited. Overall, although ordering and condensing effects characteristic to cholesterol are present in systems containing any form of CHS, their strength is appreciably weaker compared to cholesterol. Based on the considerable amount of experimental and atomistic simulation data, we conclude that these differences originate from the fact that the ester group of CHS does not anchor it in an optimal position at the water–membrane interface. The implications of these findings for considerations of protein–cholesterol interactions are briefly discussed.

Molecular Dynamics Simulation of Inverse-Phosphocholine Lipids

We have performed molecular dynamics simulations of lipid bilayers composed of inverse-phosphatidylcholines (CPe), an analog of phosphatidylcholine (PC) with the choline group directly bound to the glycerol backbone and the phosphate group freely protruding into the water phase. Synthetic phospholipids with the CPe headgroup have been proposed for use as drug delivery liposomes. Our simulation results show that the CPe lipids were characterized by a larger area per lipid molecule than the PC lipids. This can partly explain experimental results that show a higher permeability of small solutes through the membranes of liposomes composed of them. Unlike the PC headgroup, the CPe headgroup was found not to bind sodium ions at the water membrane interface. Both lipid types were found to bind calcium ions but do not bind potassium ions. Inversion of the choline group was found to decrease hydration of the membrane in the carbonyl region of the bilayer as well as hydration of the choline group. From analyzing the water ordering in our simulation, we determined that the orientation of the water layer next to the CPe membrane is effectively inverted with respect to the water ordering of the PC membrane, possibly affecting interaction with biomembranes encountered in drug delivery. Due to changes in ion binding, charge group distribution, and water orientation, the electrostatic potential profiles across the lipid bilayer of CPe membranes were found to differ considerably from those of PC membranes. This is a possible explanation of the experimentally observed changes in the charged solute permeability.

The importance of membrane defects-lessons from simulations.

The defects and pores within lipid membranes are scientifically interesting and have a number of biological applications. Although lipid bilayers are extremely thin hydrophobic barriers, just ∼3 nm thick, they include diverse chemistry and have complex structures. Bilayers are soft and dynamic, and as a result, they can bend and deform in response to different stimuli by means of structural changes in their component lipids. Though defects occur within these structures, their transience and small size have made it difficult to characterize them. However, with recent advances in computer power and computational modeling techniques, researchers can now use simulations as a powerful tool to probe the mechanism and energies of defect and pore formation in a number of situations. In this Account, we present results from our detailed molecular dynamics computer simulations of hydrophilic pores and related defects in lipid bilayers at an atomistic level. Electroporation can be used to increase the permeability of cellular membranes, with potential therapeutic applications. Atomistic simulations of electroporation have illustrated the molecular details of this process, including the importance of water dipole interactions at the water-membrane interface. Characterization of the lipid-protein interactions provides an important tool for understanding transmembrane protein structure and thermodynamic stability. Atomistic simulations give a detailed picture of the free energies of model peptides and side chains in lipid membranes; the energetic cost of defect formation strongly influences the energies of interactions between lipids and polar and charged residues. Many antimicrobial peptides form hydrophilic pores in lipid membranes, killing bacteria or cancer cells. On the basis of simulation data, at least some of these peptides form defects and pores near the center of the bilayer, with a common disordered structure where hydrated headgroups form an approximately toroidal shape. The localization and trafficking of lipids supports general membrane structure and a number of important signaling cascades, such as those involving ceramide, diacylglycerol, and cholesterol. Atomistic simulations have determined the rates and free energies of lipid flip-flop. During the flip-flop of most phosphatidylcholine lipids, a hydrophilic pore forms when the headgroup moves near the center of the bilayer. Simulations have provided novel insight into many features of defects and pores in lipid membranes. Simulation data from very different systems and models show how water penetration and defect formation can determine the free energies of many membrane processes. Bilayers can deform and allow transient defects and pores when exposed to a diverse range of stimuli. Future work will explore many aspects of membrane defects with increased resolution and scope, including the study of more complex lipid mixtures, membrane domains, and large-scale membrane remodeling. Such studies will examine processes including vesicle budding and fusion, non-bilayer lipid phases, and interactions between lipid bilayers and other biomolecules. Simulations provide information that complements experimental studies, allowing microscopic insight into experimental observations and suggesting novel hypotheses and experiments. These studies should enable a deeper understanding of the role of lipid bilayers in cellular biology and support the development of future lipid-based biotechnology.

Abstract 72: Tryptophan 415 of Scavenger Receptor BI Is Critical for Mediating Cholesterol Transport Functions

High density lipoproteins (HDL) combat atherosclerosis by delivering excess cholesteryl esters (CE) from peripheral tissues to the liver for excretion upon binding to their receptor, scavenger receptor BI (SR-BI), via reverse cholesterol transport. Our previous studies demonstrate that the extracellular domain (ECD) of SR-BI, particularly areas of hydrophobicity in its N-terminal half, is critical for cholesterol transport functions. The ECD of SR-BI also contains eight tryptophan (Trp;W) residues that may play a role in positioning SR-BI within the membrane, as observed with other membrane proteins. Based on their location at the membrane-water interface, we hypothesize that three of these Trp residue (W9, W56, W415), are critical for SR-BI’s cholesterol transport functions. To test this hypothesis, we designed single Trp-to-Phe mutations, as well as a Trp-free-SR-BI and confirmed their expression upon transient transfection into COS7 cells. Next, we used [ 3 H]/[ 125 I]-HDL-COE to assess the ability of these receptors to bind HDL. Our data revealed that Trp-free-SR-BI and W415F-SR-BI had a significantly decreased ability (88% and 63%, respectively) to bind HDL compared to wild-type (WT) SR-BI, accompanied by 44 and 64% decreases, respectively, in the ability to mediate selective uptake of HDL-COE. Trp-free- and W415F-SR-BI were also unable to enlarge the cholesterol oxidase-sensitive pool of membrane free cholesterol (FC) (20-50% decreases), as compared to WT SR-BI. The inability of these two mutant receptors to mediate these functions was not due to impaired trafficking, as both receptors were present on the cell surface as judged by flow cytometry and immunofluorescence studies. Interestingly, only Trp-free-SR-BI was unable to mediate the release of FC out of cells (50% decrease compared to WT SR-BI). Together, these data suggest that Trp415, that flanks a transmembrane segment of SR-BI, might be involved in orienting SR-BI in a conformation that would support HDL binding and selective uptake of HDL-CE, as well as reorganization of plasma membrane cholesterol. We are currently identifying the combination of Trp residues at the membrane/water interface that are important for mediating FC efflux to HDL.

Induced movements of giant vesicles by millimeter wave radiation

Our previous study of interaction between low intensity radiation at 53.37GHz and cell-size system – such as giant vesicles – indicated that a vectorial movement of vesicles was induced. This effect among others, i.e. elongation, induced diffusion of fluorescent dye di-8-ANEPPS, and increased attractions between vesicles was attributed to the action of the field on charged and dipolar residues located at the membrane–water interface. In an attempt to improve the understanding on how millimeter wave radiation (MMW) can induce this movement we report here a real time evaluation of changes induced on the movement of giant vesicles. Direct optical observations of vesicles subjected to irradiation enabled the monitoring in real time of the response of vesicles. Changes of the direction of vesicle movement are demonstrated, which occur only during irradiation with a “switch on” of the effect. This MMW-induced effect was observed at a larger extent on giant vesicles prepared with negatively charged phospholipids. The monitoring of induced-by-irradiation temperature variation and numerical dosimetry indicate that the observed effects in vesicle movement cannot be attributed to local heating.

Unknown Unknowns: the Challenge of Systematic and Statistical Error in Molecular Dynamics Simulations

Unknown Unknowns: the Challenge of Systematic and Statistical Error in Molecular Dynamics Simulations

How To Tackle the Issues in Free Energy Simulations of Long Amphiphiles Interacting with Lipid Membranes: Convergence and Local Membrane Deformations

One of the great challenges in membrane biophysics is to find a means to foster the transport of drugs across complex membrane structures. In this spirit, we elucidate methodological challenges associated with free energy computations of complex chainlike molecules across lipid membranes. As an appropriate standard molecule to this end, we consider 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled fatty amine, NBD-Cn, which is here dealt with as a homologous series with varying chain lengths. We found the membrane-water interface region to be highly sensitive to details in free energy computations. Despite considerable simulation times, we observed substantial hysteresis, the cause being the small frequency of insertion/desorption events of the amphiphile's alkyl chain in the membrane interface. The hysteresis was most pronounced when the amphiphile was pulled from water to the membrane and compromised the data that were not in line with experiments. The subtleties in umbrella sampling for computing distance along the transition path were also observed to be potential causes of artifacts. With the PGD (pull geometry distance) scheme, in which the distance from the molecule was computed to a reference plane determined by an average over all lipids in the membrane, we found marked deformations in membrane structure when the amphiphile was close to the membrane. The deformations were weaker with the PGC (pull geometry cylinder) method, where the reference plane is chosen based on lipids that are within a cylinder of radius 1.7 nm from the amphiphile. Importantly, the free energy results given by PGC were found to be qualitatively consistent with experimental data, while the PGD results were not. We conclude that with long amphiphiles there is reason for concern with regard to computations of their free energy profiles. The membrane-water interface is the region where the greatest care is warranted.

A Simple Non‐Gassing, Direct Current, Electro‐Osmotic Pump: Carbon Paper/Ceramic Frit/Carbon Paper

AbstractWe report the simplest of the small, non‐gassing, low‐voltage, direct‐current, electro‐osmotic pumps, providing enough flow for the delivery of drugs. It consists merely of a ceramic frit sandwiched between two plasma‐treated carbon paper electrodes; the plasma treatment adds electro‐oxidizable hydroquinone and electro‐reducible quinone surface functions. Protons released upon the electro‐oxidation of the hydroquinone surface functions move concertedly in the applied electric field at the ceramic membrane water interface and by dragging bulk water, causing water to flow at a rate of 10 μL cm−2 min−1 at an applied potential of 1 V across a 2 mm‐thick membrane. Following plasma treatment of the carbon paper for 45 min, the flow persists for 18 h, in which 10 mL cm−2 of water is pumped.

Defining the membrane disruption mechanism of kalata B1 via coarse-grained molecular dynamics simulations

Kalata B1 has been demonstrated to have bioactivity relating to membrane disruption. In this study, we conducted coarse-grained molecular dynamics simulations to gain further insight into kB1 bioactivity. The simulations were performed at various concentrations of kB1 to capture the overall progression of its activity. Two configurations of kB1 oligomers, termed tower-like and wall-like clusters, were detected. The conjugation between the wall-like oligomers resulted in the formation of a ring-like hollow in the kB1 cluster on the membrane surface. Our results indicated that the molecules of kB1 were trapped at the membrane-water interface. The interfacial membrane binding of kB1 induced a positive membrane curvature, and the lipids were eventually extracted from the membrane through the kB1 ring-like hollow into the space inside the kB1 cluster. These findings provide an alternative view of the mechanism of kB1 bioactivity that corresponds with the concept of an interfacial bioactivity model.

Energetic View on Membrane Pore Formation

Energetic View on Membrane Pore Formation

Site-Specific Hydration Dynamics Illuminates the Key Structural Features of Membrane-Bound α-Synuclein

α-Synuclein is an intrinsically disordered protein that is preferentially expressed in presynaptic nerve terminals. It undergoes a large-scale conformational rearrangement upon binding to synaptic vesicle membranes. In order to obtain the structural insights into the membrane-bound α-synuclein at the residue-specific resolution, we incorporated single tryptophan at various locations along the sequence. These tryptophans were used as site-specific fluorescence markers to characterize the structural and dynamical aspects of α-synuclein. The spatial localization of various parts of the protein near the membrane surface was elucidated utilizing a unique and sensitive fluorescence readout, namely, red-edge excitation shift (REES), which originates when a fluorophore is located in a highly ordered micro-environment. The extent of REES observed at different locations allowed us to directly identify the residues that are localized at the membrane-water interface comprising a thin (∼15 A) layer of motionally-constrained water molecules (Figure 1). Additionally, we have been able to distinguish subtle but important structural differences of α-synuclein bound to different lipids membranes. We believe that the structural modulations of α-synuclein on the membrane could potentially be related to its physiological functions as well as to the onset of Parkinson's diseases.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Solvent Microenvironments and Copper Binding Alters the Conformation and Toxicity of a Prion Fragment

The secondary structures of amyloidogenic proteins are largely influenced by various intra and extra cellular microenvironments and metal ions that govern cytotoxicity. The secondary structure of a prion fragment, PrP(111-126), was determined using circular dichroism (CD) spectroscopy in various microenvironments. The conformational preferences of the prion peptide fragment were examined by changing solvent conditions and pH, and by introducing external stress (sonication). These physical and chemical environments simulate various cellular components at the water-membrane interface, namely differing aqueous environments and metal chelating ions. The results show that PrP(111-126) adopts different conformations in assembled and non-assembled forms. Aging studies on the PrP(111-126) peptide fragment in aqueous buffer demonstrated a structural transition from random coil to a stable β-sheet structure. A similar, but significantly accelerated structural transition was observed upon sonication in aqueous environment. With increasing TFE concentrations, the helical content of PrP(111-126) increased persistently during the structural transition process from random coil. In aqueous SDS solution, PrP(111-126) exhibited β-sheet conformation with greater α-helical content. No significant conformational changes were observed under various pH conditions. Addition of Cu2+ ions inhibited the structural transition and fibril formation of the peptide in a cell free in vitro system. The fact that Cu2+ supplementation attenuates the fibrillar assemblies and cytotoxicity of PrP(111-126) was witnessed through structural morphology studies using AFM as well as cytotoxicity using MTT measurements. We observed negligible effects during both physical and chemical stimulation on conformation of the prion fragment in the presence of Cu2+ ions. The toxicity of PrP(111-126) to cultured astrocytes was reduced following the addition of Cu2+ ions, owing to binding affinity of copper towards histidine moiety present in the peptide.

Structural and Dynamical Insights into the Membrane-Bound α-Synuclein

Membrane-induced disorder-to-helix transition of α-synuclein, a presynaptic protein, has been implicated in a number of important neuronal functions as well as in the etiology of Parkinson’s disease. In order to obtain structural insights of membrane-bound α-synuclein at the residue-specific resolution, we took advantage of the fact that the protein is devoid of tryptophan and incorporated single tryptophan at various residue positions along the sequence. These tryptophans were used as site-specific markers to characterize the structural and dynamical aspects of α-synuclein on the negatively charged small unilamellar lipid vesicles. An array of site-specific fluorescence readouts, such as the spectral-shift, quenching efficiency and anisotropy, allowed us to discern various features of the conformational rearrangements occurring at different locations of α-synuclein on the lipid membrane. In order to define the spatial localization of various regions of the protein near the membrane surface, we utilized a unique and sensitive indicator, namely, red-edge excitation shift (REES), which originates when a fluorophore is located in a highly ordered micro-environment. The extent of REES observed at different residue positions allowed us to directly identify the residues that are localized at the membrane-water interface comprising a thin (∼ 15 Å) layer of motionally restrained water molecules and enabled us to construct a dynamic hydration map of the protein. The combination of site-specific fluorescence readouts allowed us to unravel the intriguing molecular details of α-synuclein on the lipid membrane in a direct model-free fashion. Additionally, the combination of methodologies described here are capable of distinguishing subtle but important structural alterations of α-synuclein bound to different negatively charged lipids with varied head-group chemistry. We believe that the structural modulations of α-synuclein on the membrane could potentially be related to its physiological functions as well as to the onset of Parkinson’s diseases.

Engineering Antimicrobial Peptides with Improved Antimicrobial and Hemolytic Activities

The rapid rise of antibiotic resistance in pathogens becomes a serious and growing threat to medicine and public health. Naturally occurring antimicrobial peptides (AMPs) are an important line of defense in the immune system against invading bacteria and microbial infection. In this work, we present a combined computational and experimental study of the biological activity and membrane interaction of the computationally designed Bac2A-based peptide library. We used the MARTINI coarse-grained molecular dynamics with adaptive biasing force method and the umbrella sampling technique to investigate the translocation of a total of 91 peptides with different amino acid substitutions through a mixed anionic POPE/POPG (3:1) bilayer and a neutral POPC bilayer, which mimic the bacterial inner membrane and the human red blood cell (hRBC) membrane, respectively. Potential of mean force (PMF, free energy profile) was obtained to measure the free energy barrier required to transfer the peptides from the bulk water phase to the water-membrane interface and to the bilayer interior. Different PMF profiles can indeed identify different membrane insertion scenarios by mapping out peptide-lipid energy landscapes, which are correlated with antimicrobial activity and hemolytic activity. Computationally designed peptides were further tested experimentally for their antimicrobial and hemolytic activities using bacteria growth inhibition assay and hemolysis assay. Comparison of PMF data with cell assay results reveals a good correlation of the peptides between predictive transmembrane activity and antimicrobial/hemolytic activity. Moreover, the most active mutants with the balanced substitutions of positively charged Arg and hydrophobic Trp residues at specific positions were discovered to achieve the improved antimicrobial activity while minimizing red blood cell lysis. Such substitutions provide more effective and cooperative interactions to distinguish the peptide interaction with different lipid bilayers. This work provides a useful computational tool to better understand the mechanism and energetics of membrane insertion of AMPs and to rationally design more effective AMPs.

Single Tryptophan and Tyrosine Comparisons in the N-Terminal and C-Terminal Interface Regions of Transmembrane GWALP Peptides

Hydrophobic membrane-spanning helices often are flanked by interfacial aromatic or charged residues. In this paper, we compare the consequences of single Trp → Tyr substitutions at each interface for the properties of a defined transmembrane helix in the absence of charged residues. The choice of molecular framework is critical for these single-residue experiments because the presence of "too many" aromatic residues (more than one at either membrane-water interface) introduces excess dynamic averaging of solid state NMR observables. To this end, we compare the outcomes when changing W(5) or W(19), or both of them, to tyrosine in the well-characterized transmembrane peptide acetyl-GGALW(5)(LA)6LW(19)LAGA-amide ("GWALP23"). By means of solid-state (2)H and (15)N NMR experiments, we find that Y(19)GW(5)ALP23 displays similar magnitudes of peptide helix tilt as Y(5)GW(19)ALP23 and responds similarly to changes in bilayer thickness, from DLPC to DMPC to DOPC. The presence of Y(19) changes the azimuthal rotation angle ρ (about the helix axis) to a similar extent as Y(5), but in the opposite direction. When tyrosines are substituted for both tryptophans to yield GY(5,19)ALP23, the helix tilt angle is again of comparable magnitude, and furthermore, the preferred azimuthal rotation angle ρ is relatively unchanged from that of GW(5,19)ALP23. The extent of dynamic averaging increases marginally when Tyr replaces Trp. Yet, importantly, all members of the peptide family having single Tyr or Trp residues near each interface exhibit only moderate and not highly extensive dynamic averaging. The results provide important benchmarks for evaluating conformational and dynamic control of membrane protein function.