Diphytanoyl Phosphatidylcholine Research Articles

Homogeneous hybrid droplet interface bilayers assembled from binary mixtures of DPhPC phospholipids and PB-b-PEO diblock copolymers

Hybrid membranes built from phospholipids and amphiphilic block copolymers seek to capitalize on the benefits of both constituents for constructing biomimetic interfaces with improved performance. However, hybrid membranes have not been formed or studied using the droplet interface bilayer (DIB) method, an approach that offers advantages for revealing nanoscale changes in membrane structure and mechanics and offers a path toward assembling higher-order tissues. We report on hybrid droplet interface bilayers (hDIBs) formed in hexadecane from binary mixtures of synthetic diphytanoyl phosphatidylcholine (DPhPC) lipids and low molecular weight 1,2 polybutadiene-b-polyethylene oxide (PBPEO) amphiphilic block copolymers and use electrophysiology measurements and imaging to assess the effects of PBPEO in the membrane. This work reveals that hDIBs containing up to 15 mol% PBPEO plus DPhPC are homogeneously mixtures of lipids and polymers, remain highly resistive to ion transport, and are stable—including under applied voltage. Moreover, they exhibit hydrophobic thicknesses similar to DPhPC-only bilayers, but also have significantly lower values of membrane tension. These characteristics coincide with reduced energy of adhesion between droplets and the formation of alamethicin ion channels at significantly lower threshold voltages, demonstrating that even moderate amounts of amphiphilic block copolymers in a lipid bilayer provide a route for tuning the physical properties of a biomimetic membrane.

Disentangling Memristive and Memcapacitive Effects in Droplet Interface Bilayers Using Dynamic Impedance Spectroscopy

AbstractThe underlying principles for generating intelligent behavior in living organisms are fundamentally different from those in traditional solid‐state circuits. Biomimetic neuromorphic equivalents based on biological membranes offer novel implementation of tunable plasticity and diverse mechanisms to control functionality. Here, dynamic electrochemical impedance spectroscopy (dEIS) to probe diphytanoylphosphatidylcholine (DPhPC) droplet interface bilayers (DIBs) to better understand the differences in molecular level structure/dynamics that give rise to hysteretic loops and neuromorphic, memelement behaviors in lipid bilayers in response to electrical biasing is used. Importantly, this system does not have ion‐conducting channels and is, therefore, not expected to show memristive behavior. Surprisingly, both memristive and memcapacitive behaviors by measuring the time‐dependent complex impedance of DPhPC DIBs are detected. It is shown that nonlinear memristance can originate from structural changes in the bilayer, affecting its dielectric properties. This novel dEIS application allows for the simultaneous analysis of the system's changing memristive and memcapacitive properties, which originate from different molecular restructuring processes. Moreover, and importantly, access to this type of information increases the number of neuromorphic processes supported simultaneously in a single two‐terminal device.

Stable polymer bilayers for protein channel recordings at high guanidinium chloride concentrations

The use of chaotropic reagents is common in biophysical characterization of biomolecules. When the study involves transmembrane protein channels, the stability of the protein channel and supporting bilayer membrane must be considered. In this letter, we show that planar bilayers composed of poly(1,2-butadiene)-b-poly(ethylene oxide) diblock copolymer are stable and leak-free at high guanidinium chloride concentrations, in contrast to diphytanoyl phosphatidylcholine bilayers, which exhibit deleterious leakage under similar conditions. Furthermore, insertion and functional analysis of channels such as α-hemolysin and MspA are straightforward in these polymer membranes. Finally, we demonstrate that α-hemolysin channels maintain their structural integrity at 2 M guanidinium chloride concentrations using blunt DNA hairpins as molecular reporters.

Conformation and Orientation of Branched Acyl Chains Responsible for the Physical Stability of Diphytanoylphosphatidylcholine.

Diphytanoylphosphatidylcholine (DPhPC) is a synthetic phospholipid in which two methyl-branched acyl chains are introduced into the glycerol moiety, mimicking phospholipids of eukaryotic and eubacterial origins. The lipid bilayers of DPhPC reproduce the outstanding physical properties of methyl-branched lipids that occur in archaeal membranes. DPhPC is commonly used as the base lipid in biophysical experiments, particularly for recording ion-channel currents. However, the dynamics of lipid molecules that induces their useful physical properties is still unclear. In this study, we examined the conformation and orientation of the methyl-branched acyl chain of DPhPC in a membrane using 2H nuclear magnetic resonance (NMR) measurements of the synthetic lipid with a high stereochemical purity and molecular dynamics (MD) simulations. Deuterium-labeled 3',3'-CD3,D-DPhPC (2) and 7',7'-CD3,D-DPhPC (3) showed the characteristic quadrupole splitting width in the 2H NMR spectra, which corresponded to the bent orientation reported for the archaeal lipid PGP-Me [Yamagami, M., et al. (2019) Biochemistry 58, 3869-3879]. However, MD simulations, which reproduced the 2H NMR results well, unveiled the unknown features of DPhPC in the membrane; DPhPC has a chain-specific average orientation, where two bent orientations with upward and downward methyl groups occur at positions C3 and C7 of the sn-1 and sn-2 chains of DPhPC, respectively. These MD and NMR results reveal that these two bent orientations define the average orientation of DPhPC for the shallow part of the acyl chains, which is considered to be an important factor in the stability of DPhPC membranes.

Effects of Ether Linkage on Membrane Dipole Potential and Cholesterol Flip-Flop Motion in Lipid Bilayer Membranes.

In our previous work, we investigated the effect of ether linkage on the physical properties of lipid bilayers using all-atom (AA) simulations with different water models. However, the influence of ether linkage on the transportation of cholesterol in lipid bilayers is less well studied. In order to reduce computational costs in simulations at large time and length scales, we present coarse-grained (CG) simulations of diphytanyl phosphatidylcholine (ether-DPhPC) and diphytanoyl phosphatidylcholine (ester-DPhPC) bilayer membranes in this work. First, the CG and AA simulations consistently show that the substitution of ether linkage for ester linkage would prevent the penetration of water into the lipid bilayer membranes. Second, it is encouraging that the CG simulations can nicely capture the ether effect on membrane dipole potential, showing that the ether substitution for ester would significantly decrease the dipole potential. In particular, the CG results agree with the AA simulation results, revealing that the change in the dipole potential is accompanied with the alteration in the orientation of linkage group. Finally, we carried out 60 μs CG simulations of ether-DPhPC and ester-DPhPC bilayers at two cholesterol concentrations (10 and 40% mole fraction, respectively), showing that the ether substitution for ester would facilitate the cholesterol flip-flop motion in lipid bilayer membranes.

A streptococcal lipid toxin induces membrane permeabilization and pyroptosis leading to fetal injury.

Group B streptococci (GBS) are Gram-positive bacteria that cause infections in utero and in newborns. We recently showed that the GBS pigment is hemolytic and increased pigment production promotes bacterial penetration of human placenta. However, mechanisms utilized by the hemolytic pigment to induce host cell lysis and the consequence on fetal injury are not known. Here, we show that the GBS pigment induces membrane permeability in artificial lipid bilayers and host cells. Membrane defects induced by the GBS pigment trigger K+ efflux leading to osmotic lysis of red blood cells or pyroptosis in human macrophages. Macrophages lacking the NLRP3 inflammasome recovered from pigment-induced cell damage. In a murine model of in utero infection, hyperpigmented GBS strains induced fetal injury in both an NLRP3 inflammasome-dependent and NLRP3 inflammasome-independent manner. These results demonstrate that the dual mechanism of action of the bacterial pigment/lipid toxin leading to hemolysis or pyroptosis exacerbates fetal injury and suggest that preventing both activities of the hemolytic lipid is likely critical to reduce GBS fetal injury and preterm birth.

Direct in situ measurement of specific capacitance, monolayer tension, and bilayer tension in a droplet interface bilayer.

Thickness and tension are important physical parameters of model cell membranes. However, traditional methods to measure these quantities require multiple experiments using separate equipment. This work introduces a new multi-step procedure for directly accessing in situ multiple physical properties of droplet interface bilayers (DIB), including specific capacitance (related to thickness), lipid monolayer tension in the Plateau-Gibbs border, and bilayer tension. The procedure employs a combination of mechanical manipulation of bilayer area followed by electrowetting of the capacitive interface to examine the sensitivities of bilayer capacitance to area and contact angle to voltage, respectively. These data allow for determining the specific capacitance of the membrane and surface tension of the lipid monolayer, which are then used to compute bilayer thickness and tension, respectively. The use of DIBs affords accurate optical imaging of the connected droplets in addition to electrical measurements of bilayer capacitance, and it allows for reversibly varying bilayer area. After validating the accuracy of the technique with diphytanoyl phosphatidylcholine (DPhPC) DIBs in hexadecane, the method is applied herein to quantify separately the effects on membrane thickness and tension caused by varying the solvent in which the DIB is formed and introducing cholesterol into the bilayer. Because the technique relies only on capacitance measurements and optical images to determine both thickness and tension, this approach is specifically well-suited for studying the effects of peptides, biomolecules, natural and synthetic nanoparticles, and other species that accumulate within membranes without altering bilayer conductance.

Heating-enabled formation of droplet interface bilayers using Escherichia coli total lipid extract.

Droplet interface bilayers (DIBs) serve as a convenient platform to study interactions between synthetic lipid membranes and proteins. However, a majority of DIBs have been assembled using a single lipid type, diphytanoylphosphatidylcholine (DPhPC). The work described herein establishes a new method to assemble DIBs using total lipid extract from Escherichia coli (eTLE); it is found that incubating oil-submerged aqueous droplets containing eTLE liposomes at a temperature above the gel-fluid phase transition temperature (Tg) promotes monolayer self-assembly that does not occur below Tg. Once monolayers are properly assembled via heating, droplets can be directly connected or cooled below Tg and then connected to initiate bilayer formation. This outcome contrasts immediate droplet coalescence observed upon contact between nonheated eTLE-infused droplets. Specific capacitance measurements confirm that the interface between droplets containing eTLE lipids is a lipid bilayer with thickness of 29.6 Å at 25 °C in hexadecane. We observe that bilayers formed from eTLE or DPhPC survive cooling and heating between 25 and 50 °C and demonstrate gigaohm (GΩ) membrane resistances at all temperatures tested. Additionally, we study the insertion of alamethicin peptides into both eTLE and DPhPC membranes to understand how lipid composition, temperature, and membrane phase influence ion channel formation. Like in DPhPC bilayers, alamethicin peptides in eTLE exhibit discrete, voltage-dependent gating characterized by multiple open channel conductance levels, though at significantly lower applied voltages. Cyclic voltammetry measurements of macroscopic channel currents confirm that the voltage-dependent conductance of alamethicin channels in eTLE bilayers occurs at lower voltages than in DPhPC bilayers at equivalent peptide concentrations. This result suggests that eTLE membranes, via composition, fluidity, or the presence of subdomains, offer an environment that enhances alamethicin insertion. For both membrane compositions, increasing temperature reduces the lifetimes of single channel gating events and increases the voltage required to cause an exponential increase in channel current. However, the fact that alamethicin insertion in eTLE exhibits significantly greater sensitivity to temperature changes through its Tg suggests that membrane phase plays an important role in channel formation. These effects are much less severe in DPhPC, where heating from 25 to 50 °C does not induce a phase change. The described technique for heating-assisted monolayer formation permits the use of other high transition temperature lipids in aqueous droplets for DIB formation, thereby increasing the types of lipids that can be considered for assembling model membranes.

Properties of diphytanoyl phospholipids at the air-water interface.

Diphytanoylphosphatidyl choline (DPhPC) is a synthetic ester lipid with methylated tails found in archaeal ether lipids. Because of the stability of DPhPC bilayers and the absence of phase transitions over a broad range of temperatures, the lipid is used as an artificial membrane matrix for the reconstitution of channels, pumps, and membrane-active peptides. We characterized monomolecular films made of DPhPC and its natural ether analog DOPhPC at the air-water interface. We measured compression isotherms and dipole potentials of films made of DPhPC, DPhPE, and DOPhPC. We determined that at 40 mN/m the molecular area of DPhPC is 81.2 Å(2), consistent with X-ray and neutron scattering data obtained in liposomes. This indicates that 40 mN/m is the monolayer-bilayer equivalence pressure for this lipid. At this packing density, the compressibility modulus (Cs(-1)= 122 ± 7 mN/m) and interfacial dipole potential (V = 355 ± 16 mV) were near their maximums. The molecular dipole moment was estimated to be 0.64 ± 0.02 D. The ether DOPhPC compacted to 70.4 Å(2)/lipid at 40 mN/m displaying a peak compressibility similar to that of DPhPC. The maximal dipole potential of the ether lipid was about half of that for DPhPC at this density, and the elemental dipole moment was about a quarter. The spreading of DPhPC and DOPhPC liposomes reduced the surface tension of the aqueous phase by 46 and 49 mN/m, respectively. This corresponds well to the monolayer collapse pressure. The equilibration time shortened as the temperature increased from 20 to 60 °C, but the surface pressure at equilibrium did not change. The data illustrates the properties of branched chains and the contributions of ester bonds in setting the mechanical and electrostatic parameters of diphytanoyl lipids. These properties determine an environment in which reconstituted voltage- or mechano-activated proteins may function. Electrostatic properties are important in the preparation of asymmetric folded bilayers, whereas lateral compressibility defines the tension in mechanically stimulated droplet interface bilayers.

Isoprenoid lipid chains increase membrane resistance to pore formation

The process of electroporation of bilayer lipid membranes made of diphytanoylphosphatidylcholine (DPhPC) has been studied. DPhPC combines a polar part typical for lipids of eukaryotic cells and branched isoprenoid chains forming the hydrophobic “tails” of archaea lipids. From the experimental dependence of the average lifetime on the magnitude of the applied transmembrane potential difference we evaluated line tension of the pore edge in DPhPC bilayers (Γ) and the diffusion coefficient of the defect in the space of the pore radii (D). Line tension Γ was 5.5 ± 0.2 pN and diffusion coefficient D, (6 ± 2) × 10−19 m2/s. Comparison of these values with the values typical for eukaryotic lipids suggests that the critical radius of the pore, further growth of which results in an irreversible electrical breakdown of the lipid bilayer, is less in the case of DPhPC than for conventional membranes of phosphatidylcholines, but the probability of the defect emergence and the increase of its radius is also lower for the DPhPC membranes. Apparently, these peculiarities can be accounted for by high hydrophobicity of the branched isoprenoid chains of DPhPC.

Electroporation of archaeal lipid membranes using MD simulations

Molecular dynamics (MD) simulations were used to investigate the electroporation of archaeal lipid bilayers when subjected to high transmembrane voltages induced by a charge imbalance, mimicking therefore millisecond electric pulse experiments. The structural characteristics of the bilayer, a 9:91mol% 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-myo-inositol (AI) and 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-1′(2′-O-α-d-glucosyl)-myo-inositol (AGI) were compared to small angle X-ray scattering data. A rather good agreement of the electron density profiles at temperatures of 298 and 343K was found assessing therefore the validity of the protocols and force fields used in simulations. Compared to dipalmitoyl-phosphatidylcholine (DPPC), the electroporation threshold for the bilayer was found to increase from ~2V to 4.3V at 323K, and to 5.2V at 298K. Comparing the electroporation thresholds of the archaeal lipids to those of simple diphytanoyl-phosphatidylcholine (DPhPC) bilayers (2.5V at 323K) allowed one to trace back the stability of the membranes to the structure of their lipid head groups. Addition of DPPC in amounts of 50mol% to the archaeal lipid bilayers decreases their stability and lowers the electroporation thresholds to 3.8V and 4.1V at respectively 323 and 298K. The present study therefore shows how membrane compositions can be selected to cover a wide range of responses to electric stimuli. This provides new routes for the design of liposomes that can be efficiently used as drug delivery carriers, as the selection of their composition allows one to tune in their electroporation threshold for subsequent release of their load.

Correlations of Specific Ionic Effects using Ion Channels and Surface Charge Measurements

Specific ionic effects, as captured in the Hofmeister series, have been observed in many biological phenomena including protein folding and aggregation and lipid bilayer interactions. Previously we have shown that the Hofmeister effect is present in the activity of gramicidin A channels. In particular, measurements of channel open lifetime and conductance in potassium salts clearly show the existence of two distinct ionic classes which could be identified as kosmotropic and chaotropic. To further investigate this behavior, we have measured the zeta potential of diphytanoyl phosphatidylcholine (DPhPC) liposomes in salt solutions. We observe that anions alter the surface charge of the liposomes depending on the classification of the anion as kosmotropic or chaotropic. Chaotropic anions (SCN-, ClO4-) decrease the surface charge of the liposomes while kosmotropic anions (Cl-, H2PO4-, SO42-) have the opposite effect. These results correlate with our previous studies of cation conductance through gramicidin A channels adding new insight into ionic interactions at the lipid-water interface.

Analysis of SARS-CoV E protein ion channel activity by tuning the protein and lipid charge

A partial characterization of the ion channels formed by the SARS coronavirus (CoV) envelope (E) protein was previously reported (C. Verdiá-Báguena et al., 2012 [12]). Here, we provide new significant insights on the involvement of lipids in the structure and function of the CoV E protein channel on the basis of three series of experiments. First, reversal potential measurements over a wide range of pH allow the dissection of the contributions to channel selectivity coming from ionizable residues of the protein transmembrane domain and also from the negatively charged groups of diphytanoyl phosphatidylserine (DPhPS) lipid. The corresponding effective pKas are consistent with the model pKas of the acidic residue candidates for titration. Second, the change of channel conductance with salt concentration reveals two distinct regimes (Donnan-controlled electrodiffusion and bulk-like electrodiffusion) fully compatible with the outcomes of selectivity experiments. Third, by measuring channel conductance in mixtures of neutral diphytanoyl phosphatidylcholine (DPhPC) lipids and negatively charged DPhPS lipids in low and high salt concentrations we conclude that the protein–lipid conformation in the channel is likely the same in charged and neutral lipids. Overall, the whole set of experiments supports the proteolipidic structure of SARS-CoV E channels and explains the large difference in channel conductance observed between neutral and charged membranes.

Spatial Orientation and Electric-Field-Driven Transport of Hypericin Inside of Bilayer Lipid Membranes

Fluorescence experiments were carried out to investigate the interaction of hypericin (Hyp), a natural hydrophobic photosensitizer, with artificial bilayer lipid membranes. The spatial orientation of Hyp monomers incorporated in diphytanoyl phosphatidylcholine (DPhPC) membranes was determined by measuring the dependence of the Hyp fluorescence intensity on the angle of incidence of p- and s-polarized excitation laser beams. Inside of the membrane, Hyp monomers are preferentially located in the layers near the membrane/water interface and are oriented with the S(1) ← S(0) transition dipole moments perpendicular to the membrane surface. Transport of Hyp anions between the two opposite sides of the lipid bilayer was induced by applying rectangular electric field pulses to the membrane. The characteristic time for Hyp transport through the membrane center was evaluated by the analysis of the Hyp fluorescence signal during the voltage pulses. In the zero-voltage limit, the transport time approached 70 ms and gradually decreased with higher voltage applied to the membrane. In addition, our measurements indicated an apparent pK(a) constant of 8 for Hyp deprotonation in the membrane.

The physical properties of lipid monolayers and bilayers containing calixarenes sensitive to cytochrome c

We studied physical properties of the monolayers and bilayer lipid membranes (BLM) formed by calix[6]arene carboxylic acid derivative (CX) and its mixtures with diphytanoylphosphatidylcholine (DPhPC) by means of measurement surface pressure, surface dipole potential and electrostriction. CX forms stable monolayers at an air-water interface and complexes in mixed monolayers contained DPhPC. Calixarenes increase the elastic moduli of lipid monolayers as well as BLM. Cytochrome c (cyt c) specifically binds to CX by incorporation of the amino groups of lysine residues at the protein surface. This binding affected the physical properties of CX monolayers depending on their initial surface pressure. Addition of cyt c into the water subphase induced increase of surface pressure of CX monolayers at relatively low initial pressure (15 mN/m) when monolayer was in liquid expanded state (LE). This may be due interaction of positively charged cyt c with negatively charged carboxylic groups of CX and also by its penetration into the air-water interface. However, much subtle changes were observed for higher initial surface pressure (20 and 35 mN/m) when monolayer is in liquid condensed (LC) and solid (S) state, respectively. Lysine induced substantially lower changes in surface pressure in comparison with that of cyt c.

Water Channel Formation and Ion Transport in Linear and Branched Lipid Bilayers

The composition of natural lipid membranes varies greatly depending on the type of organism, and it has been observed that small variations in lipid composition affect dramatically the membrane properties, such as structural stability and solute permeability. One of the variations is the methyl-branched lipids commonly found in archaea and bacteria. Using molecular dynamics simulations, we studied the influence of methyl branching on the electric-field induced formation of water channels in lipid bilayers and ion transports through them. We employed a double lipid bilayer setup to create within a periodic box two water compartments separated by those two bilayers. One of the compartments contains an excess of cations, while the other an equal excess of anions. This setup creates an initial transmembrane potential controlled by the ion concentration in each water compartment. We compared the response of diphytanoylphosphatidylcholine (DPhPC) lipid bilayers that have multiple methyl-branches with that of the straight-chain dipalmitoylphosphatidylcholine (DPPC) lipids. We found that compared to the straight-chain DPPC lipids, branched DPhPC lipids require a higher critical transmembrane potential and a longer time for the membrane to break down, followed by water channel formation, and transport of anions and cations through the channel. We demonstrated that while adding methyl branches reduces the lateral diffusion of the lipids, different transport properties of branched lipids are mainly due to the bulkiness of the branched lipid tails resulting in different water channel morphologies. The transmembrane potential creates toroidal pores in the straight-chain lipid bilayers, but barrel stave pores in the branched-lipid bilayers the formation of which requires a higher transmembrane potential. Our results provided a deeper understanding of the ion transport process through lipid bilayer membranes and shed light on the transport of various molecules across the lipid membranes.

Substitution of ether linkage for ester bond in phospholipids increases permeability of bilayer lipid membrane for SkQ1-type penetrating cations

Using dialkylphospholipid (diphytanyl phosphatidylcholine) instead of the conventional diacylphospholipid (diphytanoyl phosphatidylcholine) in planar lipid bilayer membranes (BLM) led to an increase in the diffusion potential of the penetrating cation plastoquinonyl-decyl-triphenylphosphonium (SkQ1), making it close to the Nernst value, and accelerated translocation of SkQ1 across the BLM as monitored by the kinetics of a decrease in the transmembrane electric current after applying a voltage (current relaxation). The consequences of changing from an ester to an ether linkage between the head groups and the hydrocarbon chains are associated with a substantial reduction in the membrane dipole potential known to originate from dipoles of tightly bound water molecules and carbonyl groups in ester bonds. The difference in the dipole potential between BLM formed of the ester phospholipid and that of the ether phospholipid was estimated to be 100 mV. In the latter case, suppression of SkQ1-mediated proton conductivity of the BLM was also observed.

Lipid dynamics and protein-lipid interactions in 2D crystals formed with the β-barrel integral membrane protein VDAC1.

We employ a combination of (13)C/(15)N magic angle spinning (MAS) NMR and (2)H NMR to study the structural and functional consequences of different membrane environments on VDAC1 and, conversely, the effect of VDAC1 on the structure of the lipid bilayer. MAS spectra reveal a well-structured VDAC1 in 2D crystals of dimyristoylphosphatidylcholine (DMPC) and diphytanoylphosphatidylcholine (DPhPC), and their temperature dependence suggests that the VDAC structure does not change conformation above and below the lipid phase transition temperature. The same data show that the N-terminus remains structured at both low and high temperatures. Importantly, functional studies based on electrophysiological measurements on these same samples show fully functional channels, even without the presence of Triton X-100 that has been found necessary for in vitro-refolded channels. (2)H solid-state NMR and differential scanning calorimetry were used to investigate the dynamics and phase behavior of the lipids within the VDAC1 2D crystals. (2)H NMR spectra indicate that the presence of protein in DMPC results in a broad lipid phase transition that is shifted from 19 to ~27 °C and show the existence of different lipid populations, consistent with the presence of both annular and bulk lipids in the functionally and structurally homogeneous samples.

A Synthetic S6 Segment Derived from KvAP Channel Self-assembles, Permeabilizes Lipid Vesicles, and Exhibits Ion Channel Activity in Bilayer Lipid Membrane

KvAP is a voltage-gated tetrameric K(+) channel with six transmembrane (S1-S6) segments in each monomer from the archaeon Aeropyrum pernix. The objective of the present investigation was to understand the plausible role of the S6 segment, which has been proposed to form the inner lining of the pore, in the membrane assembly and functional properties of KvAP channel. For this purpose, a 22-residue peptide, corresponding to the S6 transmembrane segment of KvAP (amino acids 218-239), and a scrambled peptide (S6-SCR) with rearrangement of only hydrophobic amino acids but without changing its composition were synthesized and characterized structurally and functionally. Although both peptides bound to the negatively charged phosphatidylcholine/phosphatidylglycerol model membrane with comparable affinity, significant differences were observed between these peptides in their localization, self-assembly, and aggregation properties onto this membrane. S6-SCR also exhibited reduced helical structures in SDS micelles and phosphatidylcholine/phosphatidylglycerol lipid vesicles as compared with the S6 peptide. Furthermore, the S6 peptide showed significant membrane-permeabilizing capability as evidenced by the release of calcein from the calcein-entrapped lipid vesicles, whereas S6-SCR showed much weaker efficacy. Interestingly, although the S6 peptide showed ion channel activity in the bilayer lipid membrane, despite having the same amino acid composition, S6-SCR was significantly inactive. The results demonstrated sequence-specific structural and functional properties of the S6 wild type peptide. The selected S6 segment is probably an important structural element that could play an important role in the membrane interaction, membrane assembly, and functional property of the KvAP channel.

Structure and Stability of the Spinach Aquaporin SoPIP2;1 in Detergent Micelles and Lipid Membranes

BackgroundSoPIP2;1 constitutes one of the major integral proteins in spinach leaf plasma membranes and belongs to the aquaporin family. SoPIP2;1 is a highly permeable and selective water channel that has been successfully overexpressed and purified with high yields. In order to optimize reconstitution of the purified protein into biomimetic systems, we have here for the first time characterized the structural stability of SoPIP2;1.Methodology/Principal FindingWe have characterized the protein structural stability after purification and after reconstitution into detergent micelles and proteoliposomes using circular dichroism and fluorescence spectroscopy techniques. The structure of SoPIP2;1 was analyzed either with the protein solubilized with octyl-β-D-glucopyranoside (OG) or reconstituted into lipid membranes formed by E. coli lipids, diphytanoylphosphatidylcholine (DPhPC), or reconstituted into lipid membranes formed from mixtures of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPE), 1-palmitoyl-2oleoyl-phosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS), and ergosterol. Generally, SoPIP2;1 secondary structure was found to be predominantly α-helical in accordance with crystallographic data. The protein has a high thermal structural stability in detergent solutions, with an irreversible thermal unfolding occurring at a melting temperature of 58°C. Incorporation of the protein into lipid membranes increases the structural stability as evidenced by an increased melting temperature of up to 70°C.Conclusion/SignificanceThe results of this study provide insights into SoPIP2;1 stability in various host membranes and suggest suitable choices of detergent and lipid composition for reconstitution of SoPIP2;1 into biomimetic membranes for biotechnological applications.