Tethered bilayer lipid membranes studied by simultaneous attenuated total reflectance infrared spectroscopy and electrochemical impedance spectroscopy

The formation of tethered lipid bilayer membranes (tBLMs) from unilamelar vesicles of egg yolk phosphatidylcholine (EggPC) on mixed self-assembled monolayers (SAMs) from varying ratios of 6-mercaptohexanol and EO(3)Cholesteryl on gold has been monitored by simultaneous attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS). The influence of the lipid orientation (and hence the anisotropy) of lipids on a gold film on the dichroic ratio was studied by simulations of spectra with a matrix method for anisotropic layers. It is shown that for certain tilt angles of the dielectric tensor of the adsorbed anisotropic layer dispersive and negative absorption bands are possible. The experimental data indicate that the structure of the assemblies obtained varies with varying SAM composition. On SAMs with a high content of EO(3)Cholesteryl, tBLMs with reduced fluidity are formed. For SAMs with a high content of 6-mercaptohexanol, the results are consistent with the adsorption of flattened vesicles, and spherical vesicles have been found in a small range of surface compositions. The kinetics of the adsorption process is consistent with the assumption of spherical vesicles as long-living intermediates for surfaces of a high 6-mercaptohexanol content. No long-living spherical vesicles have been detected for surfaces with a large fraction of EO(3)Cholesteryl tethers. The observed differences between the surfaces suggest that for the formation of tBLMs (unlike supported BLMs) no critical surface coverage of vesicles is needed prior to lipid bilayer formation.     

Protein deformation of lipid hybrid bilayer membranes studied by sum frequency generation vibrational spectroscopy

Structural deformations of lipid hybrid bilayer membranes induced by signal peptideless (SPL) proteins have been studied for the first time using the inherently surface specific nonlinear optical technique of sum frequency generation vibrational spectroscopy. Specifically, deformations of 1,2-distearoylphosphatidylglycerol(DSPG) membranes induced by interaction with FGF-1, a SPL protein which is released asa function of cellular stress through a nonclassical pathway, have been investigated. FGF-1 was found to induce lipid alkyl chain deformations in previously highly ordered DSPG membranes at the extremely low concentration of 1 nM at 60 degrees C. The deformation process was shown to exhibit a degree of reversibility upon removal of the protein by rinsing with buffer solution.     

Interaction between dendrons directly studied by single-molecule force spectroscopy

In this article, we have investigated the interaction between two poly(benzyl ether) dendrons directly by single-molecule force spectroscopy. For this purpose, one dendron was immobilized on an AFM tip through a poly(ethylene glycol) (PEG) spacer, and the other dendron was anchored on a gold substrate as a self-assembled monolayer. Two dendrons approached and then interacted with each other when the AFM tip and the substrate moved close together. The rupture force between dendrons was measured while the AFM tip and the substrate separated. PEG as a flexible spacer can function as a length window for recognizing the force signals and avoiding the disturbance of the interaction between the AFM tip and the substrate. The interaction between two first-generation dendrons is measured to be about 224 pN at a force loading rate of 40 nN/s. The interaction between second- and first-generation dendrons rises to 315 pN at the same loading rate. Such interactions depend on the force loading rate in the range of several to hundreds of nanonewtons per second, indicating that the rupture between dendrons is a dynamic process. The study of the interaction between surface-bound dendrons of different generations provides a model system for understanding the surface adhesion of molecules with multiple branches. In addition, this multiple-branch molecule may be used to mimic the sticky feet of geckos as a man-made adhesive.     

Phase transition of a single lipid bilayer measured by sum-frequency vibrational spectroscopy

In this communication, we demonstrate the first use of sum-frequency generation (SFG) vibrational spectroscopy to measure directly the phase transition temperature (Tm) of a single planar supported lipid bilayer (PSLB). Three saturated phospholipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (DHPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), were studied. Lipid bilayer films were prepared by the the Langmuir-Blodgett method at a surface pressure of 30 nN/m. The symmetric nature of the bilayer was used to determine the Tm of bilayers by measuring the intensity of the symmetric methyl stretch at 2875 cm-1 from the lipid fatty acid chains as a function of temperature. A maximum in the CH3 symmetric stretch transition was observed at the Tm of the lipid film due to the reduction of symmetry in the bilayer. The SFG measured Tm for DPPC, DHPC, and DSPC were 41.0 +/- 0.4, 52.4 +/- 0.7, and 57.9 +/- 0.5 degrees C, respectively. These values correlate well with the literature values of 41.3 +/- 1.8, 49 +/- 3, and 54.5 +/- 1.5 degrees C for DPPC, DHPC, and DSPC, respectively obtained by differential scanning calorimetry (DSC) of lipid vesicles in solution. The high degree of correlation between the SFG spectroscopic measurements and the DSC results suggests the Tm of these lipids is not significantly altered upon immobilization on a surface.     

Incorporation of a photosynthetic supramolecular complex by using imidazolyl Zn porphyrin dimers in bilayer lipid membrane

Incorporation of an artificial photosynthetic complex in bilayer lipid membrane by using seven porphyrin units through a supramolecular approach.     

Interfacial electron transfer dynamics in dye-modified graphene oxide nanosheets studied by single-molecule fluorescence spectroscopy

Graphene oxide (GO) nanosheets have received a great deal of attention for a wide range of applications from optoelectronic devices to biological sensors. We now report a mechanistic study of the interfacial electron transfer (ET) processes between organic dye molecule, 9-phenyl-2,3,7-trihydroxy-6-fluorone (PF), and nanometre-sized GO sheets using ensemble-averaged and single-molecule spectroscopies. The ET dynamics was characterized by the direct observation of the PF radical cation during the laser flash photolysis, and its reaction rate was determined to be ~10(11) s(-1). The single-molecule fluorescence spectroscopy was utilized to clarify the heterogeneous nature of the interfacial ET within individual composites. Their fluorescence lifetimes and spectra were found to vary from composite to composite, possibly due to the different local structures and molecular interactions. The autocorrelation analysis of fluorescence intensity trajectories also revealed the temporal fluctuation of the ET reactivity.     

Impedance spectroscopy of OmpF porin reconstituted into a mercury-supported lipid bilayer

The channel-forming protein OmpF porin was incorporated in a biomimetic membrane consisting of a lipid bilayer tethered to a mercury electrode via a thiolipid, and it was investigated in aqueous KCl by electrochemical impedance spectroscopy. The impedance spectra, recorded from 1 x 10(-2) to 1 x 10(5) Hz over a potential range of 0.7 V, were fitted to an equivalent circuit consisting of four RC meshes. The dependence of the resulting circuit elements upon the applied potential was interpreted on the basis of a general approximate approach based on a model of the electrified interphase and on the kinetics of the translocation of potassium and chloride ions across the lipid bilayer, assisted by the OmpF porin.     

Vesicle adsorption and lipid bilayer formation on glass studied by atomic force microscopy

The adsorption of phosphatidylcholine (PC) vesicles (30, 50, and 100 nm nominal diameters) and of dye-labeled PC vesicles (labeled with 6% Texas Red fluorophore (TR) and encapsulated carboxy fluorescein (CF)) to glass surfaces was studied by contact mode atomic force microscopy in aqueous buffer. These studies were performed in part to unravel details of the previously observed isolated rupture of dye-labeled PC vesicles on glass (Johnson, J. M.; Ha, T.; Chu, S.; Boxer, S. G. Biophys. J. 2002, 83, 3371-3379), specifically to differentiate partial rupture, that is, pore formation and leakage of entrapped dye, from full rupture to form bilayer disks. In addition, the adhesion potential of PC vesicles on glass was calculated based upon the adhesion-driven flattening of adsorbed vesicles and a newly developed theoretical model. The vesicles were found to flatten considerably upon adsorption to glass (width-to-height ratio of approximately 5), which leads to an estimate for the adhesion potential and for the critical rupture radius of 1.5 x 10(-4) J/m2 and 250 nm, respectively. Independent of vesicle size and loading with dye molecules, the adsorption of intact vesicles was observed at all concentrations below a threshold concentration, above which the formation of smooth lipid bilayers occurred. In conjunction with previous work (Johnson, J. M.; Ha, T.; Chu, S.; Boxer, S. G. Biophys. J. 2002, 83, 3371-3379), these data show that 6% TR 20 mM CF vesicles adsorb to the surface intact but undergo partial rupture in which they exchange content with the external buffer.     

Rydberg states of the K2 molecule studied by laser spectroscopy in a supersonic beam

Rydberg states of potassium dimer have been studied in a crossed laser-molecular beam experiment. The K₂ molecules were formed in a supersonic expansion and excited by low-power cw dye laser. Two different excitation schemes have been used: The first scheme uses a single mode ring dye laser to induce near resonant two-photon transitions while in the second scheme stepwise excitation with two dye lasers is used. In each case excitation of Rydberg levels was detected by monitoring the ionization signal resulting from three-photon absorption. We report a detailed study of 700 two-photon resonances between 625 nm and 650 nm. Most of these signals can be assigned to transitions from the X¹σ _g ⁺ to¹σ _g ⁺ ,¹Π _g , and^1Δ _g states, which are all enhanced by the B¹Π _u intermediate state. Accurate rotational constants are given for the populated vibrational levels of these states. By stepwise excitation of Rydberg levels via theB ¹Π _u state we identify 3 series of Rydberg states as¹Δ _g (4S+nD),¹Σ _g ⁺ (4S+nD), and¹Σ _g ⁺ (4S+nS) with principal quantum numbers 7≦n≦20. Molecular constants of these and other observed but as yet unidentified states are given; quantum defects and dissociation energies are discussed.     

Reduction of cytochrome c at a lipid bilayer modified electrode

The reduction of horse heart cytochrome c has been investigated at a platinum electrode modified with a lipid bilayer membrane (BLM) which immobilized vinyl ferrocene as an electron mediator. The current-voltage curves show that the direct electrochemistry of cytochrome c at the metal electrode occurs quite efficiently. An adsorption equilibrium constant for cytochrome at the BLM surface, as well as an electron transfer rate constant between the protein and the modified electrode have been estimated from these results. The values of both parameters are much higher than those reported with other types of electrode modifications, indicating that a lipid bilayer-modified platinum electrode system using vinyl ferrocene as a mediator provides substantial improvements in electrochemical activity of cytochrome c at metal electrodes. The potential for modifying and utilizing this new class of “biomembrane-like” electrode surface for metalloprotein electrochemistry is briefly discussed.     

Fluorescence spectroscopy of C60 in toluene solutions at 5 K

We have recorded the dispersed fluorescence and the fluorescence excitation spectra of C₆₀ in toluene matrices at 5 K. Upon excitation with the green Ar⁺ laser line (λ=514 nm) we obtained for the first time in this matrix well resolved visible fluorescence spectra which we have compared with those observed in other low temperature matrices. Our spectra were interpreted and assigned using theoretical assessments of vibronic activities of transitions between the three lowest excited electronic states ¹T_1g, ¹T_2g, ¹G_g and the totally symmetric ground state, and on the basis of a single 0⁰ level which has pseudo-Jahn–Teller (JT) components of the three near-degenerate excited states. The fluorescence spectra exhibit prominent JT induced h_g(1) progressions, Herzberg–Teller-induced h_u and other ungerade mode vibrations, including a very active t_1u(4) mode. Excitation wavelength independent bands are assigned to the fluorescence of C₆₀ molecules in toluene microcrystals embedded in the toluene glass whereas excitation wavelength dependent features are interpreted as originating from C₆₀ molecules isolated in the toluene glass itself. These interpretations are supported by the results of spectrally selective detected fluorescence excitation spectra.     

Sampling unfolding intermediates in calmodulin by single-molecule spectroscopy

We used single-pair fluorescence resonance energy transfer (spFRET) measurements to characterize denatured and partially denatured states of the multidomain calcium signaling protein calmodulin (CaM) in both its apo and Ca(2+)-bound forms. The results demonstrate the existence of an unfolding intermediate. A CaM mutant (CaM-T34C-T110C) was doubly labeled with fluorescent probes AlexaFlour 488 and Texas Red at opposing globular domains. Single-molecule distributions of the distance between fluorophores were obtained by spFRET at varying levels of the denaturant urea. Multiple conformational states of CaM were observed, and the amplitude of each conformation was dependent on urea concentration, with the amplitude of an extended conformation increasing upon denaturation. The distributions at intermediate urea concentrations could not be adequately described as a combination of native and denatured conformations, showing that CaM does not denature via a two-state process and demonstrating that at least one intermediate is present. The intermediate conformations formed upon addition of urea were different for Ca(2+)-CaM and apoCaM. An increase in the amplitude of a compact conformation in CaM was observed for apoCaM but not for Ca(2+)-CAM upon the addition of urea. The changes in the single-molecule distributions of CaM upon denaturation can be described by either a range of intermediate structures or by the presence of a single unfolding intermediate that grows in amplitude upon denaturation. A model for stepwise unfolding of CaM is suggested in which the domains of CaM unfold sequentially.     

Structure of a gel phase lipid bilayer prepared by the Langmuir-Blodgett/Langmuir-Schaefer method characterized by sum-frequency vibrational spectroscopy

The structure of a planar supported lipid bilayer (PSLB) prepared by the Langmuir-Blodgett (LB)/Langmuir-Schaefer (LS) method was investigated by sum-frequency vibrational spectroscopy (SFVS). By using asymmetric lipid bilayers composed of selectively deuterated 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) lipids, the orientation of the fatty acid chains and phosphocholine headgroups has been determined independently for both leaflets of the bilayer. The alkyl chains of the lipids were found to be orientated approximately 13 degrees +/- 4 degrees from the surface normal for both leaflets. The lipid chains in both leaflets also contain some gauche content, which is consistent with previous NMR and FTIR studies of similar lipid systems. More importantly, the relative number of gauche defects does not seem to be influenced by the deposition method, LB versus LS. The headgroup orientation for the lipid film in contact with the silica support was determined to be 69 degrees +/- 3 degrees , whereas that in contact with the aqueous phase was 66 degrees +/- 4 degrees from the surface normal. The SFVS results indicate that the structure of the DSPC lipid film in contact with the solid support and the film adjacent to the aqueous phase are nearly identical in structure. These results suggesting the LB/LS deposition method do indeed produce symmetric lipid bilayers. These studies further add to the growing information on the efficacy of PSLBs as suitable models for biological membrane studies.     

Modification of a supported lipid bilayer by polyelectrolyte adsorption

Addition of a weak polyelectrolyte, poly(methacrylic acid) (PMA), to a supported phospholipid bilayer made from 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) depresses the melting temperature and alters the morphology of the bilayer in the gel phase. Ellipsometry measurements show that PMA adsorption lowers the phase transition temperature by 2.4 degrees C. Atomic force microscopy (AFM) showed no visible contrast in the fluid phase (above the melting temperature) but a rich morphology in the gel phase. In the gel phase, adsorption leads to formation of significantly less mobile phospholipid islands and other defects. One consequence of this lower mobility is a decrease in the implied cooperativity number of the phase transition, N, when polymer is added. Additionally, AFM images of the gel-phase bilayer show a highly defected structure that anneals significantly more slowly than in the absence of adsorbed polymer. Tentatively, we suggest that PMA preferentially decorates island and defect edges of the DMPC bilayer.     

Dynamics of a lipid bilayer induced by electric fields

The dynamics of a lipid bilayer of 1-palmitoyl-2-oleoyl-glycero-3-phospho-ethanolamine, POPE, is investigated under the effect of two electric field intensities. The box of 720 lipids and 13,458 water molecules-plus boundary conditions-undergoes similar re-organizational dynamics in the presence of fields of 0.35 V nm(-1) and 0.5 V nm(-1). Water fingers form followed by some lipid translocation from one layer to the other. The re-organization kinetics is of the second order and is roughly 5 times faster at the higher field. The translocations may occur also upon field switch off, provided that their duration was sufficiently long. Driving few lipid translocations by a macroscopic tool, such as the electric field, appears possible.     

Identification of sugar isomers by single-molecule force spectroscopy

The nanomechanical properties of beta-galactan, a 1 --> 4 linked beta-d-galactose polysaccharide, were investigated with AFM-based single-molecule force spectroscopy. AFM captured a unique plateau at 640 pN in the force spectrogram of beta-galactan, which is significantly different than the plateau at 280 pN in the force spectrogram of amylose. Thus, our results demonstrate that force spectroscopy is able to discriminate between sugar isomers in which axial and equatorial bonds at C1 and C4 are swapped.     

Reconstitution of the voltage-gated K+ channel KAT1 in planar lipid bilayers

The single-channel current has been recorded for the voltage-gated K⁺ channel from Arabidopsis thaliana, KAT1, reconstituted in the planar bilayer lipid membrane (BLM). Channel-like current was observed between two aqueous phases after the addition of the proteoliposomes into one aqueous phase. In the potential range from 60 to 120 mV, the single-channel current was recorded and the conductance was calculated to 10.0–12.5 pS. The open channel probability increased with an increase of the applied membrane potential. These characteristics of the reconstituted channels are close to those of KAT1 reported by Hoshi et al. and Hedrich et al. with the patch clamp technique. This is the first work in which the isolated ion channel from higher plants was reconstituted in the planar BLM system.     

Reduction of cytochrome c at a lipid bilayer modified electrode

The reduction of horse heart cytochrome c has been investigated at a platinum electrode modified with a lipid bilayer membrane (BLM) which immobilized vinyl ferrocene as an electron mediator. The current—voltage curves show that the direct electrochemistry of cytochrome c at the metal electrode occurs quite efficiently. An adsorption equilibrium constant for cytochrome at the BLM surface, as well as an electron transfer rate constant between the protein and the modified electrode have been estimated from these results. The values of both parameters are much higher than those reported with other types of electrode modifications, indicating that a lipid bilayer-modified platinum electrode system using vinyl ferrocene as a mediator provides substantial improvements in electrochemical activity of cytochrome c at metal electrodes. The potential for modifying and utilizing this new class of “biomembrane-like” electrode surface for metalloprotein electrochemistry is briefly discussed.     

The analytical potential of chemoreception at bilayer lipid membranes

The potential use of the bilayer lipid membrane as an electrochemical sensor is discussed through a study of model systems known to cause increased membrane conductance. The limit of detection for amphotericin B, a molecule capable of forming membrane pores, is in the region of 1O^-9 M. The current—time profile is discussed in terms of a mechanism which involves micelle formation in the aqueous and lipid phases. Unlike previous experiments, two current maxima with time are observed for valinomycin response (limit of detection 1O^-11 M). The first transient signal is attributed to increased membrane permeability caused by a conformational change in valinomycin in the “surface” volume of the bilayer. Selective interactions at membranes and the nature of membrane responses are discussed in terms of analytical parameters.