Biomedical subjects

John C Conboy

Publications and source records attributed to John C Conboy.

14 recordsLinked to original sources

PubMed2006

Facile lipid flip-flop in a phospholipid bilayer induced by gramicidin A measured by sum-frequency vibrational spectroscopy.

The first direct experimental evidence that gramicidin A (gA), a transmembrane peptide, facilitates the translocation of unlabeled lipids in a phospholipid bilayer was obtained with sum-frequency vibrational spectroscopy (SFVS). SFVS was used to investigate the effect of gA on lipid flip-flop in a planar 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) lipid bilayer. The kinetics of lipid translocation were determined by an analysis of the SFVS intensity versus time at different temperatures in the presence of 2 mol % gA. The rate constants of DSPC flip-flop increase from 2 to 10 times relative to the pure DSPC system. The results indicate that facial lipid exchange can be induced by a hydrophobic transmembrane helix. The increase in lipid flip-flop rates is correlated to an increase in the gauche content of the lipid tails. The results suggest that membrane defects induced by the presence of integral membrane proteins may play a large role in modulating the rate of lipid flip-flop.

Biological Transport↗

PubMed2006

Changes in adsorbed fibrinogen upon conversion to fibrin.

The conversion of adsorbed fibrinogen to fibrin in the presence of the enzyme thrombin was studied using surface plasmon resonance (SPR), a quartz crystal microbalance (QCM), sum frequency generation (SFG), atomic force microscopy (AFM), and an elutability assay. Exposure of adsorbed fibrinogen to thrombin resulted in a mass loss at the surface consistent with fibrinopeptide release and conversion to fibrin. Changes in hydration upon conversion of adsorbed fibrinogen to fibrin were determined from comparisons of acoustic (QCM) and optical (SPR) mass adsorption data. Conversion to fibrin also resulted in the adsorbed layer becoming more strongly bound to the surface and more compact. The elutability of adsorbed fibrinogen by Triton X-100, studied with SPR, decreased from 90 +/- 5 to 6 +/- 2% after conversion to fibrin. The height of the adsorbed monolayer, as determined by AFM, decreased from 5.5 +/- 2.2 to 1.7 +/- 0.8 nm. We conclude that thrombin-catalyzed fibrinopeptide release triggers significant changes in fibrinogen conformation beyond peptide cleavage.

Adhesiveness↗

PubMed2005

Interfacial tension and electrocapillary measurements of the room temperature ionic liquid/aqueous interface.

The surface and aqueous interfacial tensions for a series of water-immiscible room-temperature ionic liquids (RTILs) have been measured. The RTILs used in this study were based on 1-alkyl-3-methylimidazolium cations (Cnmim, n=6, 8, 10, and 12) and bis(perfluoromethylsulfonyl)imide (BMSI) and bis(perfluoroethylsulfonyl)imide (BETI) anions. It was found that the surface tensions of the RTILs increased with an increasing cation chain length similar to the behavior of n-alkanes. Interfacial tensions of the RTILs with aqueous solutions, however, were found to decrease with the cation chain length, which has been attributed to the increased surface activity of the longer chain cations. We have also demonstrated the first use of electrocapillary measurements to study the polarizable RTIL/aqueous interfaces. From the electrocapillary data, the potential of zero charge (PZC) for these RTIL/aqueous interfaces was determined, as well as the relative surface excess charge and capacitance. The PZC was found to be dependent upon the structure of the anions and cations with PZC values ranging from -357 mV for C6mimBETI and -161 mV for C10mimBMSI. The electrocapillary results also show that the cations of the RTIL are becoming increasingly surface-active as the alkyl chain on the cation is lengthened, thereby modulating the interfacial potential.

Journal Article↗

PubMed2005

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.

Fatty Acids↗

PubMed2005

1,2-diacyl-phosphatidylcholine flip-flop measured directly by sum-frequency vibrational spectroscopy.

Sum-frequency vibrational spectroscopy (SFVS) is used to measure the intrinsic rate of lipid flip-flop for 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in planar-supported lipid bilayers (PSs). Asymmetric PSLBs were prepared using the Langmuir-Blodgett/Langmuir-Schaefer method by placing a perdeuterated lipid analog in one leaflet of the PSLB. SFVS was used to directly measure the asymmetric distribution of the native lipid within the membrane by measuring the decay in the CH3 v(s) intensity at 2875 cm(-1) with time and as a function of temperature. An average activation energy of 220 kJ/mol for the translocation of DMPC, DPPC, and DSPC was determined. A decrease in alkyl chain length resulted in a substantial increase in the rate of flip-flop manifested as an increase in the Arrhenius preexponential factor. The effect of lipid labeling was investigated by measuring the exchange of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-n,n-Dimethyl-n-(2',2',6',6'-tetramethyl-4'-piperidyl) (TEMPO-DPPC). The rate of TEMPO-DPPC flip-flop was an order-of-magnitude slower compared to DPPC. An activation energy of 79 kJ/mol was measured which is comparable to that previously measured by electron spin resonance. The results of this study illustrate how SFVS can be used to directly measure lipid flip-flop without the need for a fluorescent or spin-labeled lipid probe, which can significantly alter the rate of lipid translocation.

Algorithms↗

PubMed2005

Imaging chirality with surface second harmonic generation microscopy.

Chirality is a fundamental construct in nature which arises from an antisymmetric arrangement of atoms, molecules, or larger structures, resulting in the formation of nonsuperimposable mirror images. Bulk chiral effects can easily be measured using circular dichroism (CD) or optical rotary dispersion (ORD). However, the imaging of chirality originating from molecular surface films cannot be obtained with these linear optical methods. By using chiral second harmonic generation (C-SHG), with its inherent surface sensitivity and ability to discriminate between the symmetry of surface adsorbed species in combination with a counter-propagating optical geometry, we have developed the first nonlinear chiral microscope. In the study presented here, the intrinsic chirality of R- and S-(+)-1,1'-bi-2-naphthol (RBN, SBN) has been used to image a patterned planar supported lipid bilayer (PSLB) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) using C-SHG. Spatial resolution of the patterned PSLB is visible when either RBN or SBN is intercalated into the membrane. No image is observed when a racemic mixture of RBN and SBN is present. The C-SHG images are compared with those obtained from fluorescence microscopy to verify the C-SHG imaging technique. The results presented here demonstrate that C-SHG possesses the requisite surface selectivity and sensitivity to detect interfacial chirality and provides a direct route for the visualization of chirality originating from molecular surface films.

Lipid Bilayers↗

PubMed2005

Using the intrinsic chirality of a molecule as a label-free probe to detect molecular adsorption to a surface by second harmonic generation.

Chiral second harmonic generation (C-SHG) has been used for the label-free detection of (R)-(+)-1,1'-bi-2-naphthol (RBN) and (S)-(+)-1,1'-bi-2-naphthol (SBN) binding to planar-supported lipid bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphotidylcholine (POPC) based on the intrinsic chirality of the molecules. C-SHG adsorption isotherms of RBN and SBN reveal Langmuir adsorption behavior with binding constants of 2.7 +/- 0.2 x 10(5) M(-1) and 3.0 +/- 0.1 x 10(5) M(-1), respectively. The kinetics of RBN binding to a POPC bilayer was also measured. It was determined that the adsorption rate for RBN was 5.7 +/- 0.4 x 10(3) s(-1)M(-1) and the desorption rate was 2.1 +/- 0.8 x 10(-2) s(-1). From the kinetic data a binding constant of 2.7 +/- 1.0 x 10(5) M(-1) was calculated, which agrees well with the thermodynamic measurement. The C-SHG technique was correlated with surface tension measurements in order to determine the RBN surface excess within the POPC membrane. The maximum surface excess of RBN in a monolayer of POPC was 4.3 +/- 0.5 x 10(-11) mol cm2. Using the maximum surface excess in conjunction with the C-SHG binding data a lower limit of detection of 1.5 +/- 0.1 x 10(-13) mols cm(-2) was calculated. The results of these studies show that C-SHG is a powerful tool for the study of chiral molecular interactions at surfaces.

Adsorption↗

PubMed2004

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.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2004

Direct measurement of the transbilayer movement of phospholipids by sum-frequency vibrational spectroscopy.

The direct measurement of the transbilayer movement of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in a planar supported lipid bilayer (PSLB) at the fused silica/D2O interface was obtained with sum-frequency generation (SFG) vibrational spectroscopy. The intrinsic sensitivity of SFG to the symmetry of an interface was used to measure the asymmetric distribution of DSPC and perdeuterated DSPC (DSPC-d83) lipids in asymmetrically prepared DSPC/DSPC-d83 PSLBs. Changes in the membrane lipid composition due to exchange between leaflets was monitored by measuring the decay in the CH3 symmetric stretch intensity at 2875 cm-1 with time. The activation energy for transverse motion was determined directly from spectral relaxation measurements at several temperatures and was determined to be 206 +/- 18 kJ/mol. At room temperature (25 degrees C) the half-time of lipid flip-flop was calculated to be approximately 25 days. At 51 degrees C, only 7 degrees C below the main phase-transition temperature of DSPC, the half-time decreases to 25 min. These results have important implications for understanding the transbilayer movement of lipids in biological membranes.

Kinetics↗

PubMed2003

Optical-trapping Raman microscopy detection of single unilamellar lipid vesicles.

Raman spectra of individual unilamellar phospholipid vesicles ( approximately 0.6 microm in size) have been acquired by optical-trapping confocal Raman microscopy over the 900-3200-cm(-)(1) region. Raman scattering from the phospholipid bilayer of a single, trapped liposome could be detected, along with molecular species trapped within the vesicle. The Raman spectra of vesicles prepared from four different phosphatidylcholine lipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), could be readily distinguished by evaluating differences in the skeletal C-C and C-H stretching modes of the acyl hydrocarbon tails. These differences correlate with changes in lipid organization for different gel to liquid-crystal transition temperatures (T(m)): 41, 24, 7, and -20 degrees C for DPPC, DMPC, DLPC, and DOPC, respectively. The spectra could be acquired on the same trapped vesicle for several hours, which allowed the permeability of the bilayer to be investigated by monitoring the leakage of perchlorate anions from the vesicle. Vesicles prepared from pure DPPC or DOPC, with gel to liquid-crystal transition temperatures well above and well below room temperature, exhibited no detectable anion transfer. DLPC and DMPC vesicles permitted rapid ion transfer across the bilayer. The lengths of hydrocarbon tails were shorter in these two lipids, which could indicate that shorter chains lower the hydrophobic barrier of a membrane to ion transport. While the DMPC chains were longer than DLPC with a correspondingly higher T(m), the temperature of the experiment corresponds to the T(m) of DMPC, and domain boundaries between gel and liquid-crystal phases could contribute to high membrane permeability.

Liposomes↗

PubMed2003

Label-free chiral detection of melittin binding to a membrane.

The study presented here describes an innovative approach for the detection of surface-confined proteins using chiral second harmonic generation (C-SHG). A unique optical geometry has been employed which allows for the separation of the chiral and achiral nonlinear response. By utilizing this optical arrangement, the detection of chirality originating from melittin adsorbed to a planar supported lipid bilayer has been performed for the first time by C-SHG. Melittin binding to the membrane was monitored as a function of bulk concentration through detection of the C-SHG signal. Analysis of the C-SHG adsorption isotherms reveals Frumkin adsorption behavior with a positive interaction energy. The binding constant (Ka) obtained was determined to be (8.3 +/- 1.0) x 105 M-1. The results of these studies have far-reaching implication in the use of C-SHG for the label-free detection of protein association to surfaces and in the analysis of protein interfacial phenomena.

Amino Acid Sequence↗

PubMed2002

Quantitative measurements of recombinant HIV surface glycoprotein 120 binding to several glycosphingolipids expressed in planar supported lipid bilayers.

The interaction of recombinant HIV-1 surface glycoprotein gp120 (rgp120) with natural isolates of lactosylceramide (LacCer), glucosylceramide (GlcCer), and galactosylceramide (GalCer) has been quantitatively measured under equilibrium conditions using total internal reflection fluorescence (TIRF) spectroscopy. The binding affinity (K(a)) of rgp120 to these glycosphingolipids (GSLs), reconstituted at 5 mol % in supported planar lipid bilayers composed of 95 mol % POPC, is ca. 10(6) M(-1) for dissolved rgp120 concentrations greater than 25 nM. In contrast, at concentrations of rgp120 between 0.2 and 15 nM, rgp120 does not bind significantly to LacCer and GlcCer, but has a high affinity for GalCer with a measured K(a) value of 1.6 x 10(9) M(-1). However, protein surface coverage measurements show that this strong binding process accounts for very little of the total protein adsorbed over the entire concentration range studied. At a protein concentration of ca. 20 nM, the surface coverage is only 3% of that achieved at apparent saturation (i.e., when the protein concentration is ca. 220 nM). Thus the "high affinity" binding sites comprise only a small fraction of the total number of binding sites. Several other variables were investigated. Rgp120 binding behavior at membranes doped with alpha-hydroxygalactosylceramide (alpha-GalCer) was very similar to that observed with GalCer, showing that the presence/absence of an alpha-hydroxy moiety does not significantly affect galactosylceramide recognition. Phase segregation of GalCer, which occurs when the mole fraction of this GSL in a POPC bilayer exceeds ca. 0.1, was also investigated and showed no effect on binding affinity at low rgp120 concentrations. To investigate the influence of fatty acid chain length, GSLs with monodisperse C(18) and C(24) chain lengths, both with and without an alpha-hydroxy moiety, were synthesized, and their binding affinity to rgp120 was examined. Relative to the natural isolates (which contain a mixture of chain lengths), minimal differences were observed; thus among the compounds tested, fatty acid chain length does not affect GSL recognition. The results of this work should aid efforts to design anti-HIV-1 agents based on membrane-tethered, carbohydrate-based receptors for rgp120.

Antigens, CD↗

PubMed2002

Non-natural glycosphingolipids and structurally simpler analogues bind HIV-1 recombinant Gp120.

Interactions of recombinant gp120 (rgp120) with non-natural glycosphingolipids (GSLs) and structurally simpler analogues have been studied using a competitive adhesion assay. Conjugates of cellobiosyl ceramide and melibiosyl ceramide were synthetically prepared as water-soluble GSL analogues. These ligands were screened against a panel of biologically relevant analogues, and the results show that their interactions with rgp120 are comparable to natural cellular receptors. Glycolipid interactions with rgp120 were probed further by the synthesis and testing of structurally simpler analogues that were obtained by reductive amination of lactose, cellobiose, and melibiose with a biotinylated amino ethylene glycol moiety. RGp120 did not recognize conjugates lacking a lipid component. However, palmitoylation of the secondary amino alditols yielded compounds with comparable rgp120 affinity to the natural cellular receptor, galactosyl ceramide (GalCer). Taken together, the SAR showed that both a hydrophobic and a hydrophilic component are required for rgp120 recognition. Moreover, structural variability in the carbohydrate headgroup did not significantly alter rgp120 recognition indicating that this interaction is not highly specific.

Binding, Competitive↗

PubMedDate not supplied

Planar supported bilayer polymers formed from bis-diene lipids by Langmuir-Blodgett deposition and UV irradiation.

Substrate-supported lipid bilayers have been prepared from bis-diene functionalized phosphorylcholine (PC) lipids and polymerized by UV irradiation. The overall bilayer structure is largely preserved upon removal from water, although significant loss of material occurs from the upper leaflet of the bilayer, likely due to desorption at the air/water interface. The morphology and surface structure of the bilayer, as observed by AFM, indicate a substantially different arrangement of the lipids in the hydrated and dehydrated states, presumably due to the loss of water from the near surface region. These changes have been correlated with infrared spectral shifts sensitive to the conformation of the hydrocarbon chains. Protein adsorption studies show that rehydrated, polymerized bilayers retain a degree of resistance to BSA adsorption intermediate between model hydrophobic and fluid PC lipid bilayer surfaces. The degree of protein adsorption is correlated with desorption of material from the upper leaflet of the bilayer upon drying, which produces voids at which hydrophobically driven protein adsorption occurs.

Lipid Bilayers↗