gramicidin-a and 1-palmitoyl-2-oleoylphosphatidylcholine

Combining high-resolution imaging and electrophysiological recordings is key for various types of experimentation on lipid bilayers and ion channels. Here, we propose an integrated biosensing platform consisting of a microfluidic cartridge and a dedicated chip-holder to conduct such dual measurements on suspended lipid bilayers, in a user-friendly manner. To illustrate the potential of the integrated platform, we characterize lipid bilayers in terms of thickness and fluidity while simultaneously monitoring single ion channel currents. For that purpose, POPC lipid bilayers are supplemented with a fluorescently-tagged phospholipid (NBD-PE, 1% mol) for Fluorescence Recovery After Photobleaching (FRAP) measurements and a model ion channel (gramicidin, 1 nM). These combined measurements reveal that NBD-PE has no effect on the lipid bilayer thickness while gramicidin induces thinning of the membrane. Furthermore, the presence of gramicidin does not alter the lipid bilayer fluidity. Surprisingly, in lipid bilayers supplemented with both probes, a reduction in gramicidin open probability and lifetime is observed compared to lipid bilayers with gramicidin only, suggesting an influence of NBD-PE on the gramicidin ion function. Altogether, our proposed microfluidic biosensing platform in combination with the herein presented multi-parametric measurement scheme paves the way to explore the interdependent relationship between lipid bilayer properties and ion channel function.

Topics: Biosensing Techniques; Fluorescent Dyes; Gramicidin; Ion Channels; Lab-On-A-Chip Devices; Lipid Bilayers; Microfluidic Analytical Techniques; Microscopy, Confocal; Phosphatidylcholines; Phosphatidylethanolamines

The influence of the membrane on transmembrane proteins is central to a number of biological phenomena, notably the gating of stretch activated ion channels. Conversely, membrane proteins can influence the bilayer, leading to the stabilization of particular membrane shapes, topological changes that occur during vesicle fission and fusion, and shape-dependent protein aggregation. Continuum elastic models of the membrane have been widely used to study protein-membrane interactions. These mathematical approaches produce physically interpretable membrane shapes, energy estimates for the cost of deformation, and a snapshot of the equilibrium configuration. Moreover, elastic models are much less computationally demanding than fully atomistic and coarse-grained simulation methodologies; however, it has been argued that continuum models cannot reproduce the distortions observed in fully atomistic molecular dynamics simulations. We suggest that this failure can be overcome by using chemically and geometrically accurate representations of the protein. Here, we present a fast and reliable hybrid continuum-atomistic model that couples the protein to the membrane. We show that the model is in excellent agreement with fully atomistic simulations of the ion channel gramicidin embedded in a POPC membrane. Our continuum calculations not only reproduce the membrane distortions produced by the channel but also accurately determine the channel's orientation. Finally, we use our method to investigate the role of membrane bending around the charged voltage sensors of the transient receptor potential cation channel TRPV1. We find that membrane deformation significantly stabilizes the energy of insertion of TRPV1 by exposing charged residues on the S4 segment to solution.

Topics: Cell Membrane; Computer Simulation; Elasticity; Gramicidin; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Membrane Proteins; Models, Biological; Molecular Dynamics Simulation; Phosphatidylcholines; Surface Tension; TRPV Cation Channels

The gas-phase conformations of dimers of the channel-forming membrane peptide gramicidin A (GA), produced from isobutanol or aqueous solutions of GA-containing nanodiscs (NDs), are investigated using electrospray ionization-ion mobility separation-mass spectrometry (ESI-IMS-MS) and molecular dynamics (MD) simulations. The IMS arrival times measured for (2GA + 2Na)

Topics: Acetates; Butanols; Cell Membrane; Dimyristoylphosphatidylcholine; Gases; Gramicidin; Lipid Bilayers; Molecular Dynamics Simulation; Nanostructures; Peptides; Phosphatidylcholines; Protein Multimerization; Spectrometry, Mass, Electrospray Ionization

The structure of peptide antibiotic gramicidin A (gA) was studied in phosphatidylcholin liposomes modified by nonionic detergent Triton X-100. First, the detergent : lipid ratio at which the saturation of lipid membrane by Triton X-100 occurs (Re (sat)), was determined by light scattering. Measurements of steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene at sublytic concentrations of detergent showed that after saturation of the membrane by Triton X-100 microviscosity of lipid bilayer is reduced by 20%. The equilibrium conformational state of gA in phosphatidylcholine liposomes at Re (sat) was studied by CD spectroscopy. It was found that the conformational state of this channel-forming peptide changed crucially when Triton X-100 induced transition to more fluid membranes. The gA single-channel measurements were made with Triton X-100 containing bilayers. Tentative assignment of the channel type and gA structures was made by correlation of CD data with conductance histograms. Lipid-detergent system with variable viscosity developed in this work can be used to study the structure and folding of other membrane-active peptides.

Topics: Anti-Bacterial Agents; Cell Membrane; Detergents; Dynamic Light Scattering; Glycine max; Gramicidin; Liposomes; Membrane Fluidity; Membrane Potentials; Octoxynol; Phosphatidylcholines

Hydrophobic mismatch which is defined as the difference between the lipid hydrophobic thickness and the peptide hydrophobic length is known to be responsible in altering the lipid/protein dynamics. Gramicidin A (gA), a 15 residue β helical peptide which is well recognized to form ion conducting channels in lipid bilayer, may change its structure and function in a hydrophobic mismatched condition. We have performed molecular dynamics simulations of gA dimer in phospholipid bilayers to investigate whether or not the conversion from channel to non-channel form of gA dimer would occur under extreme negative hydrophobic mismatch. By varying the length of lipid bilayers from DLPC (1, 2-Dilauroyl-sn-glycero-3-phosphocholine) to DAPC (1, 2-Diarachidoyl-sn-glycero-3-phosphocholine), a broad range of mismatch was considered from nearly matching to extremely negative. Our simulations revealed that though the ion-channel conformation is retained by gA under a lesser mismatched situation, in extremely negative mismatched situation, in addition to bilayer thinning, the conformation of gA is changed and converted to a non-channel one. Our results demonstrate that although the channel conformation of Gramicidin A is the most stable structure, it is possible for gA to change its conformation from channel to non-channel depending upon the local environment of host bilayers.

Topics: Dimyristoylphosphatidylcholine; Gramicidin; Hydrophobic and Hydrophilic Interactions; Ion Channels; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Protein Structure, Secondary; Thermodynamics

The linear ion channel peptide gramicidin represents an excellent model for exploring the principles underlying membrane protein structure and function, especially with respect to tryptophan residues. The tryptophan residues in gramicidin channels are crucial for the structure and function of the channel. In order to test the importance of indole hydrogen bonding for the biophysical properties of gramicidin channels, we monitored the effect of N-methylation of gramicidin tryptophans, using a combination of steady state and time-resolved fluorescence approaches along with circular dichroism spectroscopy. We show here that in the absence of the hydrogen bonding ability of tryptophans, tetramethyltryptophan gramicidin (TM-gramicidin) is unable to maintain the single stranded, head-to-head dimeric channel conformation in membranes. Our results show that TM-gramicidin displays a red-shifted fluorescence emission maximum, lower red edge excitation shift (REES), and higher fluorescence intensity and lifetime, consistent with its nonchannel conformation. This is in agreement with the measured location (average depth) of the 1-methyltryptophans in TM-gramicidin using the parallax method. These results bring out the usefulness of 1-methyltryptophan as a fluorescent tool to examine the hydrogen bonding ability of tryptophans in proteins and peptides. We conclude that changes in the hydrogen bonding ability of tryptophans, along with coupled changes in peptide backbone structure induce the loss of single stranded β(6.3) helical dimer conformation. These results agree with earlier results from size-exclusion chromatography and single-channel measurements for TM-gramicidin, and confirm the importance of indole hydrogen bonding for the conformation and function of ion channels and membrane proteins.

Topics: Amino Acid Sequence; Circular Dichroism; Gramicidin; Hydrogen Bonding; Indoles; Lipid Bilayers; Models, Biological; Molecular Sequence Data; Phosphatidylcholines; Protein Multimerization; Protein Structure, Secondary; Protein Structure, Tertiary; Spectrometry, Fluorescence; Tryptophan

A single-chip electrochemical method based on impedance measurements in resonance mode has been employed to study lipid monolayer and bilayer formation on hydrophobic alkanethiolate and SiO(2) substrates, respectively. The processes were monitored by temporally resolving changes in interfacial capacitance and resistance, revealing information about the rate of formation, coverage, and defect density (quality) of the layers at saturation. The resonance-based impedance measurements were shown to reveal significant differences in the layer formation process of bilayers made from (i) positively charged lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC), (ii) neutral lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) on SiO(2), and (iii) monolayers made from POEPC on hydrophobic alkanethiolate substrates. The observed responses were represented with an equivalent circuit, suggesting that the differences primarily originate from the presence of a conductive aqueous layer between the lipid bilayers and the SiO(2). In addition, by adding the ion channel gramicidin D to bilayers supported on SiO(2), channel-mediated charge transport could be measured with high sensitivity (resolution around 1 pA).

Topics: Dielectric Spectroscopy; Gramicidin; Ion Channels; Ion Transport; Lipid Bilayers; Phosphatidylcholines; Silicon Dioxide

The effects of hydrophobic thickness and the molar phosphatidylglycerol (PG) content of lipid bilayers on the structure and membrane interaction of three cationic antimicrobial peptides were examined: aurein 2.2, aurein 2.3 (almost identical to aurein 2.2, except for a point mutation at residue 13), and a carboxy C-terminal analog of aurein 2.3. Circular dichroism results indicated that all three peptides adopt an alpha-helical structure in the presence of a 3:1 molar mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPC/DMPG), and 1:1 and 3:1 molar mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPC/POPG). Oriented circular dichroism data for three different lipid compositions showed that all three peptides were surface-adsorbed at low peptide concentrations, but were inserted into the membrane at higher peptide concentrations. The (31)P solid-state NMR data of the three peptides in the DMPC/DMPG and POPC/POPG bilayers showed that all three peptides significantly perturbed lipid headgroups, in a peptide or lipid composition-dependent manner. Differential scanning calorimetry results demonstrated that both amidated aurein peptides perturbed the overall phase structure of DMPC/DMPG bilayers, but perturbed the POPC/POPG chains less. The nature of the perturbation of DMPC/DMPG bilayers was most likely micellization, and for the POPC/POPG bilayers, distorted toroidal pores or localized membrane aggregate formation. Calcein release assay results showed that aurein peptide-induced membrane leakage was more severe in DMPC/DMPG liposomes than in POPC/POPG liposomes, and that aurein 2.2 induced higher calcein release than aurein 2.3 and aurein 2.3-COOH from 1:1 and 3:1 POPC/POPG liposomes. Finally, DiSC(3)5 assay data further delineated aurein 2.2 from the others by showing that it perturbed the lipid membranes of intact S. aureus C622 most efficiently, whereas aurein 2.3 had the same efficiency as gramicidin S, and aurein 2.3-COOH was the least efficient. Taken together, these data show that the membrane interactions of aurein peptides are affected by the hydrophobic thickness of the lipid bilayers and the PG content.

Topics: Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Anura; Benzothiazoles; Carbocyanines; Cell Membrane; Cell Membrane Permeability; Dimyristoylphosphatidylcholine; Fluoresceins; Gramicidin; Lipid Bilayers; Membrane Potentials; Phosphatidylcholines; Phosphatidylglycerols; Protein Structure, Secondary; Staphylococcus aureus

Recently, an increasing evidence accumulated for the existence of lipid microdomains, called lipid rafts, in cell membranes, which may play an important role in many important membrane-associated biological processes. Suitable model systems for studying biophysical properties of lipid rafts are lipid vesicles composed of three-component lipid mixtures, such as POPC/SM/cholesterol, which exhibit a rich phase diagram, including raft-like liquid-ordered/liquid-disordered phase coexistence regions. We explored the temperature, pressure and concentration-dependent phase behavior of such canonical model raft mixtures using the Laurdan fluorescence spectroscopic technique. Hydrostatic pressure has not only been used as a physical parameter for studying the stability and energetics of these systems, but also because high pressure is an important feature of certain natural membrane environments. We show that the liquid-disordered/liquid-ordered phase coexistence regions of POPC/SM/cholesterol model raft mixtures extends over a very wide temperature range of about 50 degrees C. Upon pressurization, an overall ordered membrane state is reached at pressures of approximately 1,000 bar at 20 degrees C, and of approximately 2,000 bar at 40 degrees C. Incorporation of 5 mol% gramicidin as a model ion channel slightly increases the overall order parameter profile in the l(o)+l(d) two-phase coexistence region, probably by selectively partitioning into l(d) domains, does not change the overall phase behavior, however. This behavior is in contrast to the effect of the peptide incorporation into simple, one-component phospholipid bilayer systems.

Topics: 2-Naphthylamine; Cholesterol; Fluorescence Polarization; Gramicidin; Laurates; Membrane Microdomains; Molecular Conformation; Peptides; Phosphatidylcholines; Pressure; Spectrometry, Fluorescence; Sphingomyelins; Temperature

The binding of the positively charged antimicrobial peptide cyclo[VKLdKVdYPLKVKLdYP] (GS14dK4) to various lipid bilayer model membranes was investigated using isothermal titration calorimetry. GS14dK4 is a diastereomeric lysine ring-size analogue of the naturally occurring antimicrobial peptide gramicidin S which exhibits enhanced antimicrobial and markedly reduced hemolytic activities compared with GS itself. Large unilamellar vesicles composed of various zwitterionic (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine [POPC]) and anionic phospholipids {1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(glycerol)] [POPG] and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phosphoserine] [POPS]}, with or without cholesterol, were used as model membrane systems. Dynamic light scattering results indicate the absence of any peptide-induced major alteration in vesicle size or vesicle fusion under our experimental conditions. The binding of GS14dK4 is significantly influenced by the surface charge density of the phospholipid bilayer and by the presence of cholesterol. Specifically, a significant reduction in the degree of binding occurs when three-fourths of the anionic lipid molecules are replaced with zwitterionic POPC molecules. No measurable binding occurs to cholesterol-containing zwitterionic vesicles, and a dramatic drop in binding is observed in the cholesterol-containing anionic POPG and POPS membranes, indicating that the presence of cholesterol markedly reduces the affinity of this peptide for phospholipid bilayers. The binding isotherms can be described quantitatively by a one-site binding model. The measured endothermic binding enthalpy (DeltaH) varies dramatically (+6.3 to +26.5 kcal/mol) and appears to be inversely related to the order of the phospholipid bilayer system. However, the negative free energy (DeltaG) of binding remains relatively constant (-8.5 to -11.5 kcal/mol) for all lipid membranes examined. The relatively small variation of negative free energy of peptide binding together with a pronounced variation of positive enthalpy produces an equally strong variation of TDeltaS (+16.2 to +35.0 kcal/mol), indicating that GS14dK4 binding to phospholipids bilayers is primarily entropy driven.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Calorimetry; Cholesterol; Drug Design; Gramicidin; Lipid Bilayers; Models, Chemical; Molecular Sequence Data; Peptides, Cyclic; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines; Phospholipids; Protein Binding; Static Electricity; Stereoisomerism; Thermodynamics; Titrimetry

We have monitored the membrane-bound channel and nonchannel conformations of gramicidin utilizing red-edge excitation shift (REES), and related fluorescence parameters. In particular, we have used fluorescence lifetime, polarization, quenching, chemical modification, and membrane penetration depth analysis in addition to REES measurements to distinguish these two conformations. Our results show that REES of gramicidin tryptophans can be effectively used to distinguish conformations of membrane-bound gramicidin. The interfacially localized tryptophans in the channel conformation display REES of 7 nm whereas the tryptophans in the nonchannel conformation exhibit REES of 2 nm which highlights the difference in their average environments in terms of localization in the membrane. This is supported by tryptophan penetration depth measurements using the parallax method and fluorescence lifetime and polarization measurements. Further differences in the average tryptophan microenvironments in the two conformations are brought out by fluorescence quenching experiments using acrylamide and chemical modification of the tryptophans by N-bromosuccinimide. In summary, we report novel fluorescence-based approaches to monitor conformations of this important ion channel peptide. Our results offer vital information on the organization and dynamics of the functionally important tryptophan residues in gramicidin.

Topics: Gramicidin; Ion Channels; Liposomes; Membranes, Artificial; Phosphatidylcholines; Protein Conformation; Spectrometry, Fluorescence; Tryptophan

The effects of the channel-forming peptide gramicidin D (gD) on the conductance and electroporation thresholds of planar bilayer lipid membranes, made of the synthetic lipid 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC), was studied. High-amplitude ( approximately 200-900 mV) rectangular voltage pulses of 15 ms duration were used to perturb the bilayers and monitor the transmembrane conductance. Electroporation voltage thresholds were found, and conductance was recorded before and after electroporation. Gramicidin was added to the system in peptide/lipid ratios of 1:10, 000, 1:500, and 1:15. The addition of gD in a ratio of 1:10,000 had no effect on electroporation, but ratios of 1:500 and 1:15 significantly increased the thresholds by 16% (p < 0.0001) and 40% (p < 0.0001), respectively. Membrane conductance before electroporation was measurable only after the addition of gD and increased monotonically as the peptide/lipid ratio increased. The effect of gD on the membrane area expansivity modulus (K) was tested using giant unilamellar vesicles (GUVs). When gD was incorporated into the vesicles in a 1:15 ratio, K increased by 110%, consistent with the increase in thresholds predicted by an electromechanical model. These findings suggest that the presence of membrane proteins may affect the electroporation of lipid bilayers by changing their mechanical properties.

Topics: Biophysical Phenomena; Biophysics; Dimerization; Electric Conductivity; Electrochemistry; Electroporation; Gramicidin; Lipid Bilayers; Phosphatidylcholines

In organic solvents gramicidin A (gA) occurs as a mixture of slowly interconverting double-stranded dimers. Membrane-spanning gA channels, in contrast, are almost exclusively single-stranded beta(6,3)-helical dimers. Based on spectroscopic evidence, it has previously been concluded that the conformational preference of gA in phospholipid bilayers varies as a function of the degree of unsaturation of the acyl chains. Double-stranded pi pi(5,6)-helical dimers predominate (over single-stranded beta(6,3)-helical dimers) in lipid bilayer membranes with polyunsaturated acyl chains. We therefore examined the characteristics of channels formed by gA in 1-palmitoyl-2-oleoylphosphatidylcholine/n-decane, 1,2-dioleoylphosphatidylcholine/n-decane, and 1,2-dilinoleoylphosphatidylcholine/n-decane bilayers. We did not observe long-lived channels that could be conducting double-stranded pi pi(5,6)-helical dimers in any of these different membrane environments. We conclude that the single-stranded beta(6,3)-helical dimer is the only conducting species in these bilayers. Somewhat surprisingly, the average channel duration and channel-forming potency of gA are increased in dilinoleoylphosphatidylcholine/n-decane bilayers compared to 1-palmitoyl-2-oleoylphosphatidylcholine/n-decane and dioleoylphosphatidylcholine/n-decane bilayers. To test for specific interactions between the aromatic side chains of gA and the acyl chains of the bilayer, we examined the properties of channels formed by gramicidin analogues in which the four tryptophan residues were replaced with naphthylalanine (gN), tyrosine (gT), and phenylalanine (gM). The results show that all of these analogue channels experience the same relative stabilization when going from dioleoylphosphatidylcholine to dilinoleoylphosphatidylcholine bilayers.

Topics: Alkanes; Amino Acid Sequence; Fatty Acids, Unsaturated; Gramicidin; Ion Channels; Lipid Bilayers; Models, Biological; Models, Structural; Molecular Sequence Data; Phosphatidylcholines; Protein Structure, Secondary; Structure-Activity Relationship

The chemical and spectroscopic properties of the new fluorescent acids all(E)-8, 10, 12, 14, 16-octadecapentaenoic acid (t-COPA) and its (8Z)-isomer (c-COPA) have been characterized in solvents of different polarity, synthetic lipid bilayers, and lipid/protein systems. These compounds are reasonably photostable in solution, present an intense UV absorption band (epsilon(350 nm) approximately 10(5) M(-1) cm(-1)) strongly overlapped by tryptophan fluorescence and their emission, centered at 470 nm, is strongly polarized (r(O) = 0.385 +/- 0.005) and decays with a major component (85%) of lifetime 23 ns and a faster minor one of lifetime 2 ns (D,L-alpha-dimyristoylphosphatidylcholine (DMPC), 15 degrees C). Both COPA isomers incorporate readily into vesicles and membranes (K(p) approximately 10(6)) and align parallel to the lipids. t-COPA distributes homogeneously between gel and fluid lipid domains and the changes in polarization accurately reflect the lipid T(m) values. From the decay of the fluorescence anisotropy in spherical bilayers of DMPC and POPC it is shown that t-COPA also correctly reflects the lipid order parameters, determined by 2H NMR techniques. Resonance energy transfer from tryptophan to the bound pentaenoic acid in serum albumin in solution, and from the tryptophan residues of gramicidin in lipid bilayers also containing the pentaenoic acid, show that this probe is a useful acceptor of protein tryptophan excitation, with R(O) values of 30-34 A.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Blood Platelets; Calorimetry; Cell Membrane; Dimyristoylphosphatidylcholine; Energy Transfer; Fatty Acids, Unsaturated; Fluorescence Polarization; Fluorescent Dyes; Gels; Gramicidin; Humans; Isomerism; Kinetics; Lipid Bilayers; Liposomes; Molecular Conformation; Phosphatidylcholines; Protein Binding; Protein Structure, Secondary; Proteins; Spectrometry, Fluorescence; Tryptophan; Ultrasonography

Rigid-limit 250-GHz electron spin resonance (FIR-ESR) spectra have been studied for a series of phosphatidylcholine spin labels (n-PC, where n = 5, 7, 10, 12, 16) in pure lipid dispersions of dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), as well as dispersions of DPPC containing the peptide gramicidin A (GA) in a 1:1 molar ratio. The enhanced g-tensor resolution of 250-GHz ESR for these spin labels permitted a careful study of the nitroxide g-tensor as a function of spin probe location and membrane composition. In particular, as the spin label is displaced from the polar head group, Azz decreases and gxx increases as they assume values typical of a nonpolar environment, appropriate for the hydrophobic alkyl chains in the case of pure lipid dispersions. The field shifts of spectral features due to changes in gxx are an order of magnitude larger than those from changes in Azz. The magnetic tensor parameters measured in the presence of GA were characteristic of a polar environment and showed only a very weak dependence of Azz and gxx on label position. These results demonstrate the significant influence of GA on the local polarity along the lipid molecule, and may reflect increased penetration of water into the alkyl chain region of the lipid in the presence of GA. The spectra from the pure lipid dispersions also exhibit a broad background signal that is most significant for 7-, 10-, and 12-PC, and is more pronounced in DPPC than in POPC. It is attributed to spin probe aggregation yielding spin exchange narrowing. The addition of GA to DPPC essentially suppressed the broad background signal observed in pure DPPC dispersions.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Biophysical Phenomena; Biophysics; Electron Spin Resonance Spectroscopy; Gramicidin; Hydrogen Bonding; Membranes, Artificial; Phosphatidylcholines; Phospholipids; Spin Labels; Water

The isothermal phase behavior of three gramicidin A'/phospholipid mixtures was investigated by an equilibrium Ca(2+)-binding technique. The phospholipid component was 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), or POPS/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at a constant mole ratio of 1/4. The bulk aqueous free Ca2+ concentration, [Ca2+]*f, in equilibrium with one or two gramicidin A'/phospholipid fluid phases and a small amount of the Ca (phosphatidylserine)2 gel phase, was measured as a function of composition at 20 degrees C by use of chromophoric high-affinity Ca2+ chelators. The coexistence of two gramicidin A'/phospholipid fluid phases was detected by an invariance in [Ca2+]*f over the range of compositions throughout which the two phases coexist. The compositions of the two coexisting phases are determined by the compositions at which the invariance in [Ca2+]*f begins and ends. With each of the gramicidin A'/phospholipid mixtures, we estimate that the composition of the gramicidin-poor phase is 0.03-0.04 mole fraction gramicidin A' and the composition of the gramicidin-rich phase is 0.13-0.14 mole fraction gramicidin A'. Characterization of these phases by low-angle X-ray diffraction revealed that, in each case, the gramicidin-poor phase is an L alpha phase and the gramicidin-rich phase is an HII phase. The isothermal phase behavior of gramicidin A'/POPC mixtures at approximately 23 degrees C, as determined by low-angle X-ray diffraction, was found to be similar to that of the other gramicidin A'/phospholipid mixtures.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Binding Sites; Calcium; Chelating Agents; Gramicidin; Kinetics; Magnetic Resonance Spectroscopy; Phosphatidylcholines; Phosphatidylserines; Phosphorus Radioisotopes; Reference Standards; X-Ray Diffraction

Evidence has been found for the existence water at the protein-lipid hydrophobic interface of the membrane proteins, gramicidin and apocytochrome C, using two related fluorescence spectroscopic approaches. The first approach exploited the fact that the presence of water in the excited state solvent cage of a fluorophore increases the rate of decay. For 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5- hexatrienyl)phenyl]ethyl]carbonyl]-3-sn-PC (DPH-PC), where the fluorophores are located in the hydrophobic core of the lipid bilayer, the introduction of gramicidin reduced the fluorescence lifetime, indicative of an increased presence of water in the bilayer. Since a high protein:lipid ratio was used, the fluorophores were forced to be adjacent to the protein hydrophobic surface, hence the presence of water in this region could be inferred. Cholesterol is known to reduce the water content of lipid bilayers and this effect was maintained at the protein-lipid interface with both gramicidin and apocytochrome C, again suggesting hydration in this region. The second approach was to use the fluorescence enhancement induced by exchanging deuterium oxide (D2O) for H2O. Both the fluorescence intensities of trimethylammonium-DPH, located in the lipid head group region, and of the gramicidin intrinsic tryptophans were greater in a D2O buffer compared with H2O, showing that the fluorophores were exposed to water in the bilayer at the protein-lipid interface. In the presence of cholesterol the fluorescence intensity ratio of D2O to H2O decreased, indicating a removal of water by the cholesterol, in keeping with the lifetime data. Altered hydration at the protein-lipid interface could affect conformation, thereby offering a new route by which membrane protein functioning may be modified.

Topics: Apoproteins; Cholesterol; Cytochrome c Group; Cytochromes c; Gramicidin; Lipid Bilayers; Membrane Lipids; Membrane Proteins; Phosphatidylcholines; Spectrometry, Fluorescence; Water