Antimicrobial peptides (AMPs) are cationic amphiphiles that comprise a key component of innate immunity. Synthetic analogues of AMPs, such as the family of phenylene ethynylene antimicrobial oligomers (AMOs), recently demonstrated broad-spectrum antimicrobial activity, but the underlying molecular mechanism is unknown. Homologues in this family can be inactive, specifically active against bacteria, or nonspecifically active against bacteria and eukaryotic cells. Using synchrotron small-angle X-ray scattering (SAXS), we show that observed antibacterial activity correlates with an AMO-induced topological transition of small unilamellar vesicles into an inverted hexagonal phase, in which hexagonal arrays of 3.4-nm water channels defined by lipid tubes are formed. Polarized and fluorescence microscopy show that AMO-treated giant unilamellar vesicles remain intact, instead of reconstructing into a bulk 3D phase, but are selectively permeable to encapsulated macromolecules that are smaller than 3.4 nm. Moreover, AMOs with different activity profiles require different minimum threshold concentrations of phosphoethanolamine (PE) lipids to reconstruct the membrane. Using ternary membrane vesicles composed of DOPG:DOPE:DOPC with a charge density fixed at typical bacterial values, we find that the inactive AMO cannot generate the inverted hexagonal phase even when DOPE completely replaces DOPC. The specifically active AMO requires a threshold ratio of DOPE:DOPC = 4:1, and the nonspecifically active AMO requires a drastically lower threshold ratio of DOPE:DOPC = 1.5:1. Since most gram-negative bacterial membranes have more PE lipids than do eukaryotic membranes, our results imply that there is a relationship between negative-curvature lipids such as PE and antimicrobial hydrophobicity that contributes to selective antimicrobial activity.

We present an experimental study of the isotropic-nematic phase transition in an aqueous mixture of charged semi-flexible rods (fd virus) and neutral polymer (Dextran). A complete phase diagram is measured as a function of ionic strength and polymer molecular weight. At high ionic strength we find that adding polymer widens the isotropic-nematic coexistence region with polymers preferentially partitioning into the isotropic phase, while at low ionic strength the added polymer has no effect on the phase transition. The nematic order parameter is determined from birefringence measurements and is found to be independent of polymer concentration (or equivalently the strength of attraction). The experimental results are compared with the existing theoretical predictions for the isotropic-nematic transition in rods with attractive interactions.

The orientational distribution function of the nematic phase of suspensions of the semiflexible rodlike virus fd is measured by x-ray diffraction as a function of concentration and ionic strength. X-ray diffraction from a single-domain nematic phase of fd is influenced by interparticle correlations at low angle, while only intraparticle scatter contributes at high angle. Consequently, the angular distribution of the scattered intensity arises from only the single-particle orientational distribution function at high angle but it also includes spatial and orientational correlations at low angle. Experimental measurements of the orientational distribution function from both the interparticle (structure factor) and intraparticle (form factor) scattering were made to test whether the correlations present in interparticle scatter influence the measurement of the single-particle orientational distribution function. It was found that the two types of scatter yield consistent values for the nematic order parameter. It was also found that x-ray diffraction is insensitive to the orientational distribution function's precise form, and the measured angular intensity distribution is described equally well by both Onsager's trial function and a Gaussian. At high ionic strength, the order parameter S of the nematic phase coexisting with the isotropic phase approaches theoretical predictions for long semiflexible rods S=0.55, but deviations from theory increase with decreasing ionic strength. The concentration dependence of the nematic order parameter also better agrees with theoretical predictions at high ionic strength indicating that electrostatic interactions have a measurable effect on the nematic order parameter. The x-ray order parameters are shown to be proportional to the measured birefringence, and the saturation birefringence of fd is determined enabling a simple, inexpensive way to measure the order parameter. Additionally, the spatial ordering of nematic fd was probed. Measurements of the nematic structure factor revealed a single large peak in contrast to nematics of rigid rods.

We report experimental measurements of the phase behavior of mixtures of thin (charged semiflexible fd virus) and thick (fd-PEG created by covalently grafting poly-(ethylene glycol) to the surface of fd virus) rods. The fd-PEG are sterically stabilized but the fd virus are charged, thus varying the ionic strength of a mixture of fd and fd-PEG varies the effective diameter of the bare fd rods, and the effective diameter ratio (d ≡ D fd-PEG /D fd ). We examine the phase diagrams of the rod mixtures and find isotropic-nematic, isotropic-nematic-nematic and nematic-nematic coexisting phases with increasing concentration. In stark contrast to predictions from earlier theoretical work, we observe a nematic-nematic coexistence region bound by a lower critical point. Moreover, we show that a rescaled Onsager-type theory for binary hard rod mixtures qualitatively describes the observed phase behavior.

The viruses studied are genetically engineered, charged, semiflexible filamentous bacteriophages that are structurally identical to M13 virus, but differ either in contour length or surface charge. While varying contour length (L) we assume the persistence length (P) remains constant, and thus we alter the rod flexibility (L/P) . Surface charge is altered both by changing solution pH and by comparing two viruses, fd and M13, which differ only by the substitution of one charged for one neutral amino acid per virus coat protein. We measure both the isotropic and cholesteric coexistence concentrations as well as the nematic order parameter after unwinding the cholesteric phase in a magnetic field as a function of rod surface charge, rod length, solution ionic strength, and solution pH . The isotropic-cholesteric transition experimental results agree semiquantitatively with theoretical predictions for semiflexible, charged rods at high ionic strength, but disagree at low ionic strength.

We examine the interaction between cytoskeletal F-actin and espin 3A, a prototypical actin bundling protein found in sensory cell microvilli, including cochlear cell stereocilia. Espin induces twist distortions in F-actin as well as facilitates bundle formation. Mutations in one of the two F-actin binding sites of espin, which have been implicated in deafness, can tune espin-actin inter-actions and radically transform the system's phase behavior. These results are compared to recent theoretical work on the general phase behavior linker-rod systems.Filamentous actin (F-actin), a biological rod-like polyelectrolyte, is a principal component of the eukaryotic cytoskeleton. The organization of F-actin is controlled predominantly by actin binding proteins (ABP's), which crosslink actin into a polymorphism of bundle and network phases [1][2][3], through a process that has recently been examined theoretically [4,5]. Espins are a recently discovered class of ABP's responsible for the formation of parallel actin bundles in vivo and in vitro [6][7][8]. A specific isoform, espin 3A, is found in actin bundles in sensory cell microvilli, such as the stereocilia of cochlear hair cells, which are vital for the transduction of sound in hearing [9][10][11]. Genetic mutations in espin's F-actin binding sites are linked to malformed stereocilia, deafness and vestibular dysfunction in humans and mice [11][12][13]. Such 'deafness' mutations allow us to assess the role of linker 'stickiness' in the organization of actin bundles.In this Letter we describe the first measurements of self-assembled espin-actin bundle structure, and examine how interactions between F-actin and different espin linkers are expressed in the system's phase behavior. Small angle x-ray scattering (SAXS) shows that as espin concentration increases, the system evolves directly from an isotropic phase to hexagonally coordinated paracrystalline bundles of hyper-twisted filaments. As the ability of a given espin to crosslink F-actin is decreased by using two different genetically modified 'deafness' mutants of espin with progressively more severe damage to one of the two actin binding sites, an unanticipated nematic actin-espin network is observed. By mixing wild type (wt) and mutant espin, which allows us to continuously tune this crosslinking affinity between espin and actin (as well as mimic heterozygous expression of mutant espins), we find that the onset of this nematic-network phase is a strong function of espin crosslinking affinity and concentration. We hypothesize that this unexpected isotropic to nematic network transition, which has strong implications for stereocilia formation and hearing, is due to the way weak espin linkers crosslink actin rods. F-actin was prepared from lyophilized rabbit skeletal muscle G-actin monomer (42 kDa, Cytoskeleton, Inc). G-actin was resuspended in 5 mM Tris, with 0.2 mM CaCl 2 , 0.5 mM ATP, 0.2 mM Dithotreithol (DTT) and 0.01% NaN 3 at pH 8.0 and polymerized into F-actin upon the addition of 100 mM KCl. F-actin is t...

The fundamental nature of the nematic-nematic ͑N-N͒ phase separation in binary mixtures of rigid hard rods is analyzed within the Onsager second-virial theory and the extension of Parsons and Lee which includes a treatment of the higher-body contributions. The particles of each component are modeled as hard spherocylinders of different diameter ͑D 1 D 2 ͒, but equal length ͑L 1 = L 2 = L͒. In the case of a system which is restricted to be fully aligned ͑parallel rods͒, we provide an analytical solution for the spinodal boundary for the limit of stability of N-N demixing; only a single region of N-N coexistence bounded at lower pressures ͑densities͒ by a N-N critical point is possible for such a system. The full numerical solution with the Parsons-Lee extension also indicates that, depending on the length of the particles, there is a range of values of the diameter ratio ͑d = D 2 / D 1 ͒ where the N-N phase coexistence is closed off by a critical point at lower pressure. A second region of N-N coexistence can be found at even lower pressures for certain values of the parameters; this region is bounded by an "upper" N-N critical point. The two N-N coexistence regions can also merge to give a single region of N-N coexistence extending to very high pressure without a critical point. By including the higher-order contributions to the excluded volume ͑end effects͒ in the Onsager theory, we prove analytically that the existence of the N-N lower critical point is a direct consequence of the finite size of the particles. A new analytical equation of state is derived for the nematic phase using the Gaussian approximation. In the case of Onsager limit ͑infinite aspect ratio͒, we show that the N-N phase behavior obtained using the Parsons-Lee approach substantially deviates from that with the Onsager theory for the N-N transition due to the nonvanishing third and higher order virial coefficients. We also provide a detailed discussion of the N-N phase behavior of recent experimental results for mixtures of thin and thick rods of the same length, for which the Onsager and Parsons-Lee theories can provide a qualitative description.

We examine mixtures of DNA and filamentous actin (F-actin) as a model system of like-charged rigid rods and flexible chains. Confocal microscopy reveals the formation of elongated nematic F-actin domains reticulated via defect-free vertices into a network embedded in a mesh of random DNA. Synchrotron x-ray scattering results indicate that the DNA mesh squeezes the F-actin domains into a nematic state with an interactin spacing that decreases with increasing DNA concentration as d(actin) proportional, variantrho(DNA)(-1/2). Interestingly, the system changes from a counterion-controlled regime to a depletion-controlled regime with added salt, with drastic consequences for the osmotic pressure induced phase behavior.