Specific Electrostatic Interactions Research Articles

Uncovering Specific Electrostatic Interactions in the Denatured States of Proteins

The stability and folding of proteins are modulated by energetically significant interactions in the denatured state that is in equilibrium with the native state. These interactions remain largely invisible to current experimental techniques, however, due to the sparse population and conformational heterogeneity of the denatured-state ensemble under folding conditions. Molecular dynamics simulations using physics-based force fields can in principle offer atomistic details of the denatured state. However, practical applications are plagued with the lack of rigorous means to validate microscopic information and deficiencies in force fields and solvent models. This study presents a method based on coupled titration and molecular dynamics sampling of the denatured state starting from the extended sequence under native conditions. The resulting denatured-state pKas allow for the prediction of experimental observables such as pH- and mutation-induced stability changes. I show the capability and use of the method by investigating the electrostatic interactions in the denatured states of wild-type and K12M mutant of NTL9 protein. This study shows that the major errors in electrostatics can be identified by validating the titration properties of the fragment peptides derived from the sequence of the intact protein. Consistent with experimental evidence, our simulations show a significantly depressed pKa for Asp8 in the denatured state of wild-type, which is due to a nonnative interaction between Asp8 and Lys12. Interestingly, the simulation also shows a nonnative interaction between Asp8 and Glu48 in the denatured state of the mutant. I believe the presented method is general and can be applied to extract and validate microscopic electrostatics of the entire folding energy landscape.

Theoretical Investigation of Thermal Decomposition of Peroxidized Coelenterazines with and without External Perturbations

Thermal decomposition of peroxidized coelenterazines with and without external perturbations has been studied theoretically using the hybrid density functional theory (B3LYP) and the Coulomb-attenuating method (CAM). Possible roles of a hydrogen-bonding interface constituted by amino acid residues in the coelenterazine-biding site of aequorin are addressed by using simple model clusters with a polarizable continuum model to grasp some important aspects that may affect the electronic mechanism operating within the photoprotein. Calculations have revealed that the electronic property and stability of the peroxide are greatly affected by its protonation state and/or environmental effects, such as a polarizing medium and specific (localized) short-range electrostatic interactions, which may be critical for the bioluminescence activity. Theory highlights two mechanisms by which the neutral species can be activated, which otherwise decomposes by a homolytic O-O dissociation with a high barrier. In the first mechanism, the Tyr82-His16-Trp86 triad motif facilitates the deprotonation process of the phenolic OH group at the C(6) position of the coelenterazine and thereby makes it a sufficiently good electron donor to activate the O-O bond. In the second mechanism, intramolecular charge transfer is accomplished within the neutral peroxide by a proton delivery mediated via another triad motif, Tyr184-His169-Trp173, without the activation of the substrate itself. The combination of the first and second mechanisms leads to complete electron transfer for the formation of a radical pair as a local intermediate stabilized by the nearby triad motifs.

6-s-cis Conformation and Polar Binding Pocket of the Retinal Chromophore in the Photoactivated State of Rhodopsin

The visual pigment rhodopsin is unique among the G protein-coupled receptors in having an 11-cis retinal chromophore covalently bound to the protein through a protonated Schiff base linkage. The chromophore locks the visual receptor in an inactive conformation through specific steric and electrostatic interactions. This efficient inverse agonist is rapidly converted to an agonist, the unprotonated Schiff base of all-trans retinal, upon light activation. Here, we use magic angle spinning NMR spectroscopy to obtain the (13)C chemical shifts (C5-C20) of the all-trans retinylidene chromophore and the (15)N chemical shift of the Schiff base nitrogen in the active metarhodopsin II intermediate. The retinal chemical shifts are sensitive to the conformation of the chromophore and its molecular interactions within the protein-binding site. Comparison of the retinal chemical shifts in metarhodopsin II with those of retinal model compounds reveals that the Schiff base environment is polar. In particular, the (13)C15 and (15)Nepsilon chemical shifts indicate that the C horizontal lineN bond is highly polarized in a manner that would facilitate Schiff base hydrolysis. We show that a strong perturbation of the retinal (13)C12 chemical shift observed in rhodopsin is reduced in wild-type metarhodopsin II and in the E181Q mutant of rhodopsin. On the basis of the T(1) relaxation time of the retinal (13)C18 methyl group and the conjugated retinal (13)C5 and (13)C8 chemical shifts, we have determined that the conformation of the retinal C6-C7 single bond connecting the beta-ionone ring and the retinylidene chain is 6-s-cis in both the inactive and the active states of rhodopsin. These results are discussed within the general framework of ligand-activated G protein-coupled receptors.

Multiple intermediate states precede pore block during N-type inactivation of a voltage-gated potassium channel

N-type inactivation of voltage-gated potassium channels is an autoinhibitory process that occurs when the N terminus binds within the channel pore and blocks conduction. N-type inactivation and recovery occur with single-exponential kinetics, consistent with a single-step reaction where binding and block occur simultaneously. However, recent structure–function studies have suggested the presence of a preinactivated state whose formation and loss regulate inactivation and recovery kinetics. Our studies on N-type inactivation of the Shaker-type AKv1 channel support a multiple-step inactivation process involving a series of conformational changes in distinct regions of the N terminus that we have named the polar, flex, and latch regions. The highly charged polar region forms interactions with the surface of the channel leading up to the side window openings between the T1 domain and the channel transmembrane domains, before the rate-limiting step occurs. This binding culminates with a specific electrostatic interaction between R18 and EDE161-163 located at the entrance to the side windows. The latch region appears to work together with the flex region to block the pore after polar region binding occurs. Analysis of tail currents for a latch region mutant shows that both blocked and unblocked states exist after the rate-limiting transition is passed. Our results suggest that at least two intermediate states exist for N-type inactivation: a polar region–bound state that is formed before the rate-limiting step, and a pre-block state that is formed by the flex and latch regions during the rate-limiting step.

Architectural Underpinnings of the Genetic Code for Glutamine

Structure-based mutational analysis was used to probe the architecture of the glutamine binding pocket in Escherichia coli glutaminyl-tRNA synthetase (GlnRS). Crystallographic studies of several different GlnRS complexes in a lattice that supports catalytic activity have shown that the glutamine amide group makes only ambiguous hydrogen-bonding interactions with a tyrosine hydroxyl and bound water molecule, rather than the highly specific hydrogen-bonding and electrostatic interactions made by the substrate amino acid in all other nonediting tRNA synthetases. Further, the amide oxygen of substrate glutamine accepts a hydrogen bond from the 3'-ribose hydroxyl group of ATP, an unusual distal substrate-substrate interaction also not observed in any other tRNA synthetase complex. Steady-state and pre-steady-state kinetic analysis using a 3'-dATP analogue in place of ATP shows that removal of this distal interaction does not affect K(m) for the analogue as compared with ATP, yet decreases the efficiency of aminoacylation by 10(3)-fold while significantly elevating K(m) for glutamine. In other experiments, mutation of eight nearly fully conserved residues in the glutamine binding pocket reveals decreases in k(cat)/K(m) ranging from 5- to 400-fold, and in K(d) for glutamine of up to at least 60-fold. Amino acid replacements at Tyr211 and Gln255, which participate with substrate glutamine in an antidromic circular arrangement of hydrogen bonds, cause the most severe decreases in catalytic efficiency. This finding suggests that the relative absence of direct hydrogen bonds to glutamine may be in part compensated by additional binding energy derived from the enhanced stability of this circular network. Calculations of electrostatic surface potential in the active site further suggest that a complementary electrostatic environment is also an important determinant of glutamine binding.

Engineering and validation of a novel lipid thin film for biomembrane modeling in lipophilicity determination of drugs and xenobiotics

BackgroundDetermination of lipophilicity as a tool for predicting pharmacokinetic molecular behavior is limited by the predictive power of available experimental models of the biomembrane. There is current interest, therefore, in models that accurately simulate the biomembrane structure and function. A novel bio-device; a lipid thin film, was engineered as an alternative approach to the previous use of hydrocarbon thin films in biomembrane modeling.ResultsRetention behavior of four structurally diverse model compounds; 4-amino-3,5-dinitrobenzoic acid (ADBA), naproxen (NPX), nabumetone (NBT) and halofantrine (HF), representing 4 broad classes of varying molecular polarities and aqueous solubility behavior, was investigated on the lipid film, liquid paraffin, and octadecylsilane layers. Computational, thermodynamic and image analysis confirms the peculiar amphiphilic configuration of the lipid film. Effect of solute-type, layer-type and variables interactions on retention behavior was delineated by 2-way analysis of variance (ANOVA) and quantitative structure property relationships (QSPR). Validation of the lipid film was implemented by statistical correlation of a unique chromatographic metric with Log P (octanol/water) and several calculated molecular descriptors of bulk and solubility properties.ConclusionThe lipid film signifies a biomimetic artificial biological interface capable of both hydrophobic and specific electrostatic interactions. It captures the hydrophilic-lipophilic balance (HLB) in the determination of lipophilicity of molecules unlike the pure hydrocarbon film of the prior art. The potentials and performance of the bio-device gives the promise of its utility as a predictive analytic tool for early-stage drug discovery science.

“Barbed nanowires” from polymers via electrospinning

AbstractElectrospinning is a highly versatile technique that allows producing fibers with diameters down to a few nanometers not only from polymers but also from metals, metal oxides, or ceramics. Fiber formation in electrospinning differs strongly from other fiber producing methods such as extrusion in that it is basically governed by self‐assembly processes induced by specific electrostatic interactions following the Earnshaw theorem of electrostatics. This allows the production of nanofibers with very peculiar shapes. Here, we report the one step fabrication of barbed nanofibers due to a particular choice of the spinning conditions. Such barbed fibers allow, among others, to control the total porosity of nanofiber nonwovens and to reduce the tendency of linear nano‐objects towards aggregation. POLYM. ENG. SCI., 2009. © 2008 Society of Plastics Engineers

Synergistic effect of basic residues at positions 14–15 of nociceptin on binding affinity and receptor activation

Nociceptin is an endogenous ligand that activates a G protein-coupled receptor ORL1 and contains two indispensable Arg-Lys (RK) dipeptide units at positions 8–9 and 12–13. By replacing an additional RK unit at positions 6–7, 10–11, 14–15, or 16–17, of the peptide we have identified the analog, [RK 14–15]nociceptin as a superagonist. In fact, this peptide exhibits 3-fold higher binding affinity and 17-fold greater potency in a functional GTPγS-binding assay compared to wild-type nociceptin. Here, we have further investigated the role of basic residues in position 14–15. The replacement of three other possible basic dipeptides, KR, RR, and KK, into nociceptin at positions 14–15 resulted in similar enhancements of binding affinity (3–5-fold) and biological potency (10–12-fold in the GTPγS assay). However, when only a single basic residue (Arg or Lys) was replaced in either position 14 or 15, all the resulting analogs showed moderate enhancements of binding and biological activity (2–4-fold in both). These results indicate that the addition of basic charges in positions 14 and 15 enhance in a synergistic fashion the interaction of nociceptin with the receptor and only the simultaneous presence of two adjacent basic residues yields an optimal effect. This suggests that specific electrostatic interactions between both amino acids present in 14–15 and corresponding residues in the receptor are responsible for the enhancement of nociceptin activity.

Fabrication, structure, and properties of chitin whisker‐reinforced alginate nanocomposite fibers

AbstractCalcium alginate yarn (30 fibers) and calcium alginate nanocomposite yarn (30 fibers) containing 0.05–2.00% w/w chitin whiskers were both prepared by wet spinning process. The whiskers were prepared by acid hydrolysis of chitin from shrimp shells. The average length and width of the whiskers were 343 and 46 nm, with the aspect ratio being ∼ 7.5. Incorporation of a low amount of the whiskers in the nanocomposite fibers improved both the mechanical and the thermal properties of the fibers significantly, possibly a result of the specific interactions, i.e., hydrogen bonding and electrostatic interactions, between the alginate molecules and the homogeneously dispersed chitin whiskers. Biodegradation of the calcium alginate fibers and the nanocomposite fibers was tested in Tris‐HCl buffer solution and the same buffer solution that contained lysozyme. The addition of the chitin whiskers in the nanocomposites fibers accelerated the biodegradation process of the fibers in the presence of lysozyme, whereas the presence of Ca2+ ions in the Tris‐HCl buffer solution helped to improve the tenacity of both the alginate and the nanocomposite fibers. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

Interaction of Zinc Tetrasulfonated Phthalocyanine with Cytochrome c in Water and Triton-X 100 Micelles

The interaction of a zinc tetrasulfonated phthalocyanine with cytochrome c was studied using steady-state spectroscopic techniques and time-correlated single photon counting in water and Triton-X 100 micelles. The dye forms dimers in water with a high equilibrium constant (70 x 10(6) M(-1)). Because of a specific electrostatic interaction, the presence of cytochrome c does not lead to a dissociation of this dimer, but increases its formation, with an equilibrium constant of about 7.9 x 10(9) M(-1). Triton-X 100 micelles dissociate the dimer, creating two populations of dye molecules: one in a hydrophilic media, probably on the surface of the micelles, another on a hydrophobic environment, probably inside the micelles. However, when cytochrome c is added the dye aggregation is again induced leading to a strong fluorescence quenching. This fluorescence quenching may also be caused by a photoinduced electron-transfer due to the formation of a 1:1 complex between the dye and the protein, but the present work does not give direct evidence of such an effect because the fluorescence decays did not show the presence of an extra component. The results presented here are quite different from those reported for aluminum sulfonated phthalocyanines, where aggregation does not occur and the fluorescence quenching is solely due to photoinduced electron-transfer reactions.

Binding to human serum albumin of zidovudine (AZT) and novel AZT derivatives. Experimental and theoretical analyses☆

This work presents the binding of AZT and nine novel AZT derivatives to human serum albumin (HSA), both defatted (HSA(D)) and complexed with fatty acids (HSA(FA)). The bound fractions and binding site were determined by applying an ultrafiltration procedure, with an increased affinity for the majority of these derivatives to HSA(D) being found with respect to that of AZT, while only one derivative exhibited an increased affinity for HSA(FA). By means of computational methods, we observed that specific electrostatic interactions are responsible for the increased affinity for HSA(D), while the presence of fatty acids complexed to HSA caused an intense electrostatic repulsion with negatively charged ligands located in Sudlow site I, thus diminishing their bound fractions. A strong relationship between the calculated energetic components and the observed experimental affinity was identified.

Structure of a Complex of Human Lactoferrin N-lobe with Pneumococcal Surface Protein A Provides Insight into Microbial Defense Mechanism

Human lactoferrin, a component of the innate immune system, kills a wide variety of microorganisms including the Gram positive bacteria Streptococcus pneumoniae. Pneumococcal surface protein A (PspA) efficiently inhibits this bactericidal action. The crystal structure of a complex of the lactoferrin-binding domain of PspA with the N-lobe of human lactoferrin reveals direct and specific interactions between the negatively charged surface of PspA helices and the highly cationic lactoferricin moiety of lactoferrin. Binding of PspA blocks surface accessibility of this bactericidal peptide preventing it from penetrating the bacterial membrane. Results of site-directed mutagenesis, in vitro protein binding assays and isothermal titration calorimetry measurements corroborate that the specific electrostatic interactions observed in the crystal structure represent major associations between PspA and lactoferrin. The structure provides a snapshot of the protective mechanism utilized by pathogens against the host's first line of defense. PspA represents a major virulence factor and a promising vaccine candidate. Insights from the structure of the complex have implications for designing therapeutic strategies for treatment and prevention of pneumococcal diseases that remain a major public health problem worldwide.

Preparation and Characterization of Innovative Protein-coated Poly(Methylmethacrylate) Core-shell Nanoparticles for Vaccine Purposes

This study aims at developing novel core-shell poly(methylmethacrylate) (PMMA) nanoparticles as a delivery system for protein vaccine candidates. Anionic nanoparticles consisting of a core of PMMA and a shell deriving from Eudragit L100/55 were prepared by an innovative synthetic method based on emulsion polymerization. The formed nanoparticles were characterized for size, surface charge and ability to reversibly bind two basic model proteins (Lysozyme, Trypsin) and a vaccine relevant antigen (HIV-1 Tat), by means of cell-free studies. Their in vitro toxicity and capability to preserve the biological activity of the HIV-1 Tat protein were studied in cell culture systems. Finally, their safety and immunogenicity were investigated in the mouse model. The nanoparticles had smooth surface, spherical shape and uniform size distribution with a mean diameter of 220 nm. The shell is characterized by covalently bound carboxyl groups negatively charged at physiological pH, able to reversibly adsorb large amounts (up to 20% w/w) of basic proteins (Lysozyme, Trypsin and HIV-1 Tat), mainly through specific electrostatic interactions. The nanoparticles were stable, not toxic to the cells, protected the HIV-1 Tat protein from oxidation, thus preserving its biological activity and increasing its shelf-life, and efficiently delivered and released it intracellularly. In vivo experiments showed that they are well tolerated and elicit strong immune responses against the delivered antigen in mice. This study demonstrates that these new nanoparticles provide a versatile platform for protein surface adsorption and a promising delivery system particularly when the maintenance of the biologically active conformation is required for vaccine efficacy.

The Specificity in the Interaction between Cytochrome f and Plastocyanin from the Cyanobacterium Nostoc sp. PCC 7119 Is Mainly Determined by the Copper Protein

The plastocyanin-cytochrome f complex from Nostoc exhibits relevant structural differences when compared with the homologous complexes from other cyanobacteria and plants, with electrostatic and hydrophobic interactions being differently involved in each case. Here, five negatively charged residues of a recombinant form of cytochrome f from Nostoc have been replaced with either neutral or positively charged residues, and the effects of mutations on the kinetics of electron transfer to wild-type and mutant forms of plastocyanin have been measured by laser flash absorption spectroscopy. Cytochrome f mutants with some negative charges replaced with neutral residues exhibit an apparent electron transfer rate constant with wild-type plastocyanin similar to or slightly higher than that of the wild-type species, whereas the mutants with negative charges replaced with positive residues exhibit a significantly lower reactivity. Taken together, these results indicate that the effects of neutralizing residues at the electrostatically charged patch of cytochrome f are smaller than those previously observed for mutants of plastocyanin, thus suggesting that it is the copper protein which determines the specificity of the electrostatic interaction with the heme protein. Moreover, cross reactions between mutants of both proteins reveal the presence of some short-range specific electrostatic interactions. Our findings also make evident the fact that in Nostoc the main contribution to the electrostatic nature of the complex is provided by the small domain of cytochrome f.

Miscibility and mechanical properties of quaternized polysulfone/benzoyl guar gum blends

Novel blends from quaternized polysulfone (QPSF) and benzoyl guar gum (BGG) coded as QB with different contents (10–80wt%) were prepared through solution casting method. Simultaneously, other kinds of blends were prepared from chloromethylated polysulfone (ClPSF) and BGG coded as ClB to compare the effects of the substituted groups on the miscibility and properties of the composite materials. The effect of BGG content on QB blends was investigated by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), atom force microscopy (AFM), differential scanning calorimetry (DSC) and tensile tests. The results revealed that QB blends had good or certain miscibility over the entire composition ratio of BGG to QPSF under study. Compared with ClB blends, QB blends exhibited stronger interfacial attraction and better phase mixing as a result of the relatively strong hydrogen bonding and the specific electrostatic interaction between QPSF and BGG. The occurrence of strong interaction between QPSF and BGG played a key role in improving the material performance. With an increase of BGG content in the blends, the tensile strength of QB blends increased from 33.1 to 44.3MPa. Furthermore, the mechanical properties of QB-20 blend at different RH were also discussed. It was found that the composite properties changed considerably with moisture content, which attributed that water molecules had a great effect on the hydrogen bonding between the two polymers.

The retention properties of nucleobases in alkyl C 8-/C 18- and IAM-chromatographic systems in relation to log Pow

In order to evaluate the differences in the partition properties of 35 structurally congeneric nucleobases of biological interests in octanol–water biphasic, alkyl C 8/C 18, and IAM systems, a comparative chromatographic study was performed. Comparing with the reversed-phase C 8/C 18 retention data, most of the purines possessed weaker IAM retention except for those with specific H-bond and/or electrostatic interactions. Quantitative correlations between the experimental log P ow literature values and the IAM, C 8, and C 18 log k were evaluated ( R 2 = 0.943, 0.794, and 0.767, respectively). Although IAM retention correlated significantly better (larger R 2 value) with the log P ow values statistically, the latter was revealed apparently behaving more like (slope approaching unity) alkyl C 8/C 18 retention and hence also has the same shortcoming in under-representing analytes capable of forming short-term H-bond/electrostatic interactions with polar head-groups of phospholipids. A chemically meaningful structure-retention model ( q 2 = 0.824 and R 2 = 0.968) was derived, in which the hydrophobic interaction is identified as the underlying factor for the retention of purines in IAM system modulated non-trivially by H-bond/electrostatic interactions.

Protein imaging on a semiconducting substrate: A scanning tunnelling microscopy investigation

The reverse transcriptase (RT) enzymes of the human immune deficiency virus 1 (HIV-1) and of the avian myeloblastosis virus (AMV) are deposited onto the semiconducting substrate n-MoTe2 and imaged by scanning tunnelling microscopy (STM). Molecule selection was made after considering the unique complex and specific enzyme structures and medical relevance. Protein immobilization is attributed to specific substrate structural defects and localized electrostatic interaction. The ultrastructure resolution obtained is ascribed to energetic and charge transfer properties of metal tip/protein/semiconductor contacts with an energetic shift of semiconductor band edges due to (partial) Fermi level pinning and/or formation and reversal of a strong inversion layer. Protein electron transport combining Poole–Frenkel-type transport in insulators and solvation-induced electron detrapping is suggested.

Thermochromism and thermofluorochromism of merocyanines with a positive solvatochromism

The thermochromism and thermofluorochromism of ethanol solutions of di-, tetra-, and hexamethine merocyanines based on 1,3-dihydro-1,3,3-trimethyl-(2H)-indol-2-yliden and malonodinitrile, having a positive solvatochromism as compared to the corresponding cationic and anionic symmetric dyes, are studied at T = 348, 293, and 4.2 K. It is found that, upon a decrease in the temperature, the absorption spectra exhibit bathochromic shifts of bands, an increase in the vinylene shifts, and a decrease in deviations, whereas the fluorescence spectra exhibit opposite changes. In this case, the differences in the shapes of the absorption and luminescence bands decrease so considerably that the bands become virtually mirror-symmetric. It is shown that the spectral and luminescent effects observed arise because, at high temperatures, the electronic structure of the ground state of merocyanines approaches that of neutral polyenes, whereas, at low temperatures, this structure is similar to the structure of ideal polymethine. Such a transformation of the structure is caused by an increase in specific electrostatic interactions (nucleophilic and electrophilic solvations) with a solvent, as a result of which the alternation of the charges of a chromophore increases, while its bond orders become equalized.

The barrier for proton transport in aquaporins as a challenge for electrostatic models: The role of protein relaxation in mutational calculations

The origin of the barrier for proton transport through the aquaporin channel is a problem of general interest. It is becoming increasingly clear that this barrier is not attributable to the orientation of the water molecules across the channel but rather to the electrostatic penalty for moving the proton charge to the center of the channel. However, the reason for the high electrostatic barrier is still rather controversial. It has been argued by some workers that the barrier is due to the so-called NPA motif and/or to the helix macrodipole or to other specific elements. However, our works indicated that the main reason for the high barrier is the loss of the generalized solvation upon moving the proton charge from the bulk to the center of the channel and that this does not reflect a specific repulsive electrostatic interaction but the absence of sufficient electrostatic stabilization. At this stage it seems that the elucidation and clarification of the origin of the electrostatic barrier can serve as an instructive test case for electrostatic models. Thus, we reexamine the free-energy surface for proton transport in aquaporins using the microscopic free-energy perturbation/umbrella sampling (FEP/US) and the empirical valence bond/umbrella sampling (EVB/US) methods as well as the semimacroscopic protein dipole Langevin dipole model in its linear response approximation version (the PDLD/S-LRA). These extensive studies help to clarify the nature of the barrier and to establish the "reduced solvation effect" as the primary source of this barrier. That is, it is found that the barrier is associated with the loss of the generalized solvation energy (which includes of course all electrostatic effects) upon moving the proton charge from the bulk solvent to the center of the channel. It is also demonstrated that the residues in the NPA region and the helix dipole cannot be considered as the main reasons for the electrostatic barrier. Furthermore, our microscopic and semimacroscopic studies clarify the problems with incomplete alternative calculations, illustrating that the effects of various electrostatic elements are drastically overestimated by macroscopic calculations that use a low dielectric constant and do not consider the protein reorganization. Similarly, it is pointed out that microscopic potential of mean force calculations that do not evaluate the electrostatic barrier relative to the bulk water cannot be used to establish the origin of the electrostatic barrier. The relationship between the present study and calculations of pK(a)s in protein interiors is clarified, pointing out that approaches that are applied to study the aquaporin barrier should be validated by pK(a)s calculations. Such calculations also help to clarify the crucial role of solvation energies in establishing the barrier in aquaporins.

Mechanisms of antimicrobial peptide action: Studies of indolicidin assembly at model membrane interfaces by in situ atomic force microscopy

We report here on an in situ atomic force microscopy study of the interaction of indolicidin, a tryptophan-rich antimicrobial peptide, with phase-segregated zwitterionic DOPC/DSPC supported planar bilayers. By varying the peptide concentration and bilayer composition through the inclusion of anionic lipids (DOPG or DSPG), we found that indolicidin interacts with these model membranes in one of two concentration-dependent manners. At low peptide concentrations, indolicidin forms an amorphous layer on the fluid domains when these domains contain anionic lipids. At high peptide concentrations, indolicidin appears to initiate a lowering of the gel-phase domains independent of the presence of an anionic lipid. Similar studies performed using membrane-raft mimetic bilayers comprising 30 mol% cholesterol/1:1 DOPC/egg sphingomyelin revealed that indolicidin does not form a carpet-like layer on the zwitterionic DOPC domains at low peptide concentrations and does not induce membrane lowering of the liquid-ordered sphingomyelin/cholesterol-rich domains at high peptide concentration. Simultaneous AFM–confocal microscopy imaging did however reveal that indolicidin preferentially inserts into the fluid-phase DOPC domains. These data suggest that the indolicidin–membrane association is influenced greatly by specific electrostatic interactions, lipid fluidity, and peptide concentration. These insights provide a glimpse into the mechanism of the membrane selectivity of antibacterial peptides and suggest a powerful correlated approach for characterizing peptide–membrane interactions.