Ming-Tao Lee

Evidence for Membrane Thinning Effect as the Mechanism for Peptide-Induced Pore Formation

Antimicrobial peptides have two binding states in a lipid bilayer, a surface state S and a pore-forming state I. The transition from the S state to the I state has a sigmoidal peptide-concentration dependence indicating cooperativity in the peptide-membrane interactions. In a previous paper, we reported the transition of alamethicin measured in three bilayer conditions. The data were explained by a free energy that took into account the membrane thinning effect induced by the peptides. In this paper, the full implications of the free energy were tested by including another type of peptide, melittin, that forms toroidal pores, instead of barrel-stave pores as in the case of alamethicin. The S-to-I transitions were measured by oriented circular dichroism. The membrane thinning effect was measured by x-ray diffraction. All data were in good agreement with the theory, indicating that the membrane thinning effect is a plausible mechanism for the peptide-induced pore formations.

Mechanism and kinetics of pore formation in membranes by water-soluble amphipathic peptides

How antimicrobial peptides form pores in membranes is of interest as a fundamental membrane process. However, the underlying molecular mechanism, which has potential applications in therapeutics, nonviral gene transfer, and drug delivery, has been in dispute. We have resolved this mechanism by observing the time-dependent process of pore formation in individual giant unilamellar vesicles (GUVs) exposed to a melittin solution. An individual GUV first expanded its surface area at constant volume and then suddenly reversed to expanding its volume at constant area. The area expansion, the volume expansion, and the point of reversal all match the results of equilibrium measurements performed on peptide–lipid mixtures. The mechanism includes a negative feedback that makes peptide-induced pores stable with a well defined size, contrary to the suggestion that peptides disintegrate the membrane in a detergent-like manner.

Sigmoidal concentration dependence of antimicrobial peptide activities: A case study on alamethicin

The transition of the state of alamethicin from its inactive state to its active state of pore formation was measured as a function of the peptide concentration in three different membrane conditions. In each case the fraction of the alamethicin molecules occupying the active state, phi, showed a sigmoidal concentration dependence that is typical of the activities of antimicrobial peptides. Such a concentration dependence is often interpreted as due to peptide aggregation. However, we will show that a simple effect of aggregation cannot explain the data. We will introduce a model based on the elasticity of membrane, taking into consideration the membrane-thinning effect due to protein inclusion. The elastic energy of membrane provides an additional driving force for aggregation. The model produces a relation that not only predicts the correct concentration dependence but also explains qualitatively how the dependence changes with membrane conditions. The result shows that the membrane-mediated interactions between monomers and aggregates are essential for the strong cooperativity shown in pore formation.

Process of inducing pores in membranes by melittin

Melittin is a prototype of the ubiquitous antimicrobial peptides that induce pores in membranes. It is commonly used as a molecular device for membrane permeabilization. Even at concentrations in the nanomolar range, melittin can induce transient pores that allow transmembrane conduction of atomic ions but not leakage of glucose or larger molecules. At micromolar concentrations, melittin induces stable pores allowing transmembrane leakage of molecules up to tens of kilodaltons, corresponding to its antimicrobial activities. Despite extensive studies, aspects of the molecular mechanism for pore formation remain unclear. To clarify the mechanism, one must know the states of the melittin-bound membrane before and after the process. By correlating experiments using giant unilamellar vesicles with those of peptide-lipid multilayers, we found that melittin bound on the vesicle translocated and redistributed to both sides of the membrane before the formation of stable pores. Furthermore, stable pores are formed only above a critical peptide-to-lipid ratio. The initial states for transient and stable pores are different, which implies different mechanisms at low and high peptide concentrations. To determine the lipidic structure of the pore, the pores in peptide–lipid multilayers were induced to form a lattice and examined by anomalous X-ray diffraction. The electron density distribution of lipid labels shows that the pore is formed by merging of two interfaces through a hole. The molecular property of melittin is such that it adsorbs strongly to the bilayer interface. Pore formation can be viewed as the bilayer adopting a lipid configuration to accommodate its excessive interfacial area.

A small/wide-angle X-ray scattering instrument for structural characterization of air-liquid interfaces, thin films and bulk specimens

At the National Synchrotron Radiation Research Center, a small/wide-angle X-ray scattering (SAXS/WAXS) instrument has been installed at the BL23A beamline with a superconducting wiggler insertion device. This beamline is equipped with double Si(111) crystal and double Mo/B4C multilayer monochromators, and an Si-based plane mirror that can selectively deflect the beam downwards for grazing-incidence SAXS (GISAXS) studies of air–liquid or liquid–liquid interfaces. The SAXS/WAXS instrument, situated in an experimental hutch, comprises collimation, sample and post-sample stages. Pinholes and slits have been incorporated into the beam collimation system spanning a distance of ∼5 m. The sample stage can accommodate various sample geometries for air–liquid interfaces, thin films, and solution and solid samples. The post-sample section consists of a 1 m WAXS section with two linear gas detectors, a vacuum bellows (1–4 m), a two-beamstop system and the SAXS detector system, all situated on a motorized optical bench for motion in six degrees of freedom. In particular, the vacuum bellows of a large inner diameter (260 mm) provides continuous changes of the sample-to-detector distance under vacuum. Synchronized SAXS and WAXS measurements are realized via a data-acquisition protocol that can integrate the two linear gas detectors for WAXS and the area detector for SAXS (gas type or Mar165 CCD); the protocol also incorporates sample changing and temperature control for programmable data collection. The performance of the instrument is illustrated via several different measurements, including (1) simultaneous SAXS/WAXS and differential scanning calorimetry for polymer crystallization, (2) structural evolution with a large ordering spacing of ∼250 nm in a supramolecular complex, (3) SAXS for polymer blends under in situ drawing, (4) SAXS and anomalous SAXS for unilamellar lipid vesicles and metalloprotein solutions, (5) anomalous GISAXS for oriented membranes of Br-labeled lipids embedded with peptides, and (6) GISAXS for silicate films formed in situ at the air–water interface.

Membrane-Thinning Effect of Curcumin

Interaction of curcumin with lipid bilayers is not well understood. A recent experiment showed that curcumin significantly affected the single-channel lifetime of gramicidin in a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayer without affecting its single-channel conductance. We performed two experiments to understand this result. By isothermal titration calorimetry, we measured the partition coefficient of curcumin binding to DOPC bilayers. By x-ray lamellar diffraction, we measured the thickness change of DOPC bilayers as a function of the curcumin/lipid ratio. A nonlinear membrane-thinning effect by curcumin was discovered. The gramicidin data were qualitatively interpreted by the combination of isothermal titration calorimetry and x-ray results. We show that not only does curcumin thin the lipid bilayer, it might also weaken its elasticity moduli. The result implies that curcumin may affect the function of membrane proteins by modifying the properties of the host membrane.

The Bound States of Amphipathic Drugs in Lipid Bilayers: Study of Curcumin

Drug-membrane interactions are well known but poorly understood. Here we describe dual measurements of membrane thickness change and membrane area change due to the binding of the amphipathic drug curcumin. The combined results allowed us to analyze the binding states of a drug to lipid bilayers, one on the water-membrane interface and another in the hydrocarbon region of the bilayer. The transition between the two states is strongly affected by the elastic energy of membrane thinning (or, equivalently, area stretching) caused by interfacial binding. The data are well described by a two-state model including this elastic energy. The binding of curcumin follows a common pattern of amphipathic peptides binding to membranes, suggesting that the binding states of curcumin are typical for amphipathic drugs.

An instrument for time-resolved and anomalous simultaneous small- and wide-angle X-ray scattering (SWAXS) at NSRRC

A SWAXS (small- and wide-angle X-ray scattering) instrument was recently installed at the wiggler beamline BL17B3 of the National Synchrotron Radiation Research Center (NSRRC), Taiwan. The instrument, which is designed for studies of static and dynamic nanostructures and correlations between the nano (or meso) structure (SAXS) and crystalline structure (WAXS), provides a flux of 1010–1011 photon s−1 at the sample at energies between 5 and 14 keV. With a SAXS area detector and a WAXS linear detector connected to two data acquisition systems operated in master–slave mode, the instrument allows one to perform time-resolved as well as anomalous scattering measurements. Data reduction algorithms have been developed for rapid processing of the large SWAXS data sets collected during time-resolved measurements. The performance of the instrument is illustrated by examples taken from different classes of ongoing projects: (i) time-resolved SAXS/WAXS/differential scanning calorimetry (DSC) with a time resolution of 10 s on a semicrystalline poly(hexamethylene terephthalate) sample, (ii) anomalous SAXS/WAXS measurements on a nanoparticulate PtRu catalyst, and (iii) grazing-incidence SAXS of a monolayer of oriented semiconductor quantum wires, and humidity-controlled ordering of Alamethicin peptides embedded in an oriented lipid membrane.

Formation of Ge quantum dots array in layer-cake technique for advanced photovoltaics.

We report a simple and manageable growth method for placing dense three-dimensional Ge quantum dot (QD) arrays in a uniform or a graded size distribution, based on thermally oxidizing stacked poly-SiGe in a layer-cake technique. The QD size and spatial density in each stack can be modulated by conditions of the Ge content in poly-Si(1-x)Ge(x), oxidation, and the underlay buffer layer. Size-dependent internal structure, strain, and photoluminescence properties of Ge QDs are systematically investigated. Optimization of the processing conditions could be carried out for producing dense Ge QD arrays to maximize photovoltaic efficiency.

Peptide-induced bilayer thinning structure of unilamellar vesicles and the related binding behavior as revealed by X-ray scattering

We have studied the bilayer thinning structure of unilamellar vesicles (ULV) of a phospholipid 1,2-dierucoyl-sn-glycero-3-phosphocholine (di22:1PC) upon binding of melittin, a water-soluble amphipathic peptide. Successive thinning of the ULV bilayers with increasing peptide concentration was monitored via small-angle X-ray scattering (SAXS). Results suggest that the two leaflets of the ULV of closed bilayers are perturbed and thinned asymmetrically upon free peptide binding, in contrast to the centro-symmetric bilayer thinning of the substrate-oriented multilamellar membranes (MLM) with premixed melittin. Moreover, thinning of the melittin-ULV bilayer associates closely with peptide concentration in solution and saturates at ~4%, compared to the ~8% maximum thinning observed for the correspondingly premixed peptide-MLM bilayers. Linearly scaling the thinning of peptide-ULV bilayers to that of the corresponding peptide-MLM of a calibrated peptide-to-lipid ratio, we have deduced the number of bound peptides on the ULV bilayers as a function of free peptide concentration in solution. The hence derived X-ray-based binding isotherm allows extraction of a low binding constant of melittin to the ULV bilayers, on the basis of surface partition equilibrium and the Gouy-Chapman theory. Moreover, we show that the ULV and MLM bilayers of di22:1PC share a same thinning constant upon binding of a hydrophobic peptide alamethicin; this result supports the linear scaling approach used in the melittin-ULV bilayer thinning for thermodynamic binding parameters of water-soluble peptides.