Biomedical subjects

Per Lincoln

Publications and source records attributed to Per Lincoln.

At least 19 recordsLinked to original sources

PubMed2007

Binding geometry and photophysical properties of DNA-threading binuclear ruthenium complexes.

The DNA binding conformation and the photophysical properties of the semiflexible binuclear ruthenium complex [micro-bidppz(phen)4Ru2]4+ (2) were studied with optical spectroscopy and compared to the rigid, planar homologue in syn conformation [micro-dtpf(phen)4Ru2]4+ (3) and the parent "light-switch" complex [Ru(phen)2dppz]2+ (1). Comparison of calculated and observed absorption bands of the bridging ligand, bidppz, confirm earlier suggestions that 2 is significantly nonplanar, both free in solution and when intercalated into poly(dAdT)2, but the conclusion that the intercalated conformation is an anti rotamer is not substantiated by comparison of linear and circular dichroism spectra of 2 and 3. The behavior of the emission quantum yield as a function of temperature is similar for the two binuclear complexes 2 and 3 in different protic solvents, and a quantitative analysis suggests that, in solution, the solvent is more strongly hydrogen bonded to the excited state of 2 than to 1. However, the observation that for 2 the radiative rate constant increases to a value similar to 1 upon intercalation into DNA suggests that the difference between 1 and 2 in accepting hydrogen bonds is less pronounced when intercalated.

Chlorides↗

PubMed2006

Controlling desensitized states in ligand-receptor interaction studies with cyclic scanning patch-clamp protocols.

Ligand-gated ion channels are important control elements in regulation of cellular activities, and increasing evidence demonstrates their role as therapeutic targets. The receptors display complex desensitization kinetics, occurring on vastly different time scales. This is not only important in biology and pharmacology but might also be of technological significance since populations of receptors under microfluidic control can function analogously to DRAM memory circuits. Using a novel microfluidic method, and computer modeling of the receptor state distributions, we here demonstrate that GABAA receptor populations can be controlled to display high or low EC50 values, depending on input function (i.e., the exact pattern of agonist application). The sensitivity of the receptors can be tuned up to 40-fold (beta-alanine) by the particular agonist exposure pattern. By combining patch-clamp experiments with computer modeling of receptor state distributions, we can control the assembly of receptors in desensitized states. The technique described can be used as an analytical tool to study the effect of desensitization on the activity of ion channel effectors. We describe the differential blocking effect of the competitive antagonist bicuculline on the high- and low-EC50 GABAA receptor preparations and conclude that the inhibition is dramatically dependent on how the different desensitized states are populated. Furthermore, we show that both GABA and beta-alanine, two agonists with different affinity but similar efficacy, induce the same type of desensitization behavior and memory effects in GABAA receptors.

Animals↗

PubMed2006

Membrane interactions of cell-penetrating peptides probed by tryptophan fluorescence and dichroism techniques: correlations of structure to cellular uptake.

This work reports on the binding and conformation of a series of CPPs in the bilayer membranes of large unilamellar vesicles and the effect of the presence of cholesterol. We show a negative correlation between alpha-helical structure and uptake efficiency for penetratin peptides where the two central arginine residues of penetratin are thought to be important for breaking the secondary structure. Penetratin alpha-helicity is also reduced upon incorporation of cholesterol into the membrane. Flow linear dichroism in the far-UV region shows that the penetratin peptides adopt a preferential orientation of the alpha-helix parallel to the bilayer, and the linear dichroism (LD) spectrum in the aromatic region indicates that the tryptophan residues are preferentially oriented parallel to the membrane. The Tat analogue TatP59W and the oligoarginine R7W, which are more efficient CPPs than penetratin, bind to membranes as random coils and do not show any orientation in LD, again indicating that alpha-helicity reduces uptake efficiency. Further, we observe large variations in tryptophan quantum yields for the five CPPs in this study and discuss this in terms of the ability to cause lipid rearrangement. Binding isotherms show that cholesterol increases the affinity of the peptide for the membrane, but tryptophan fluorescence lifetimes are essentially unaltered by incorporation of as much as 40 mol % cholesterol into the membrane, suggesting the absence of specific peptide-cholesterol interactions. Fluorescence emission maxima are insensitive to cholesterol and indicate that the peptide is positioned in the headgroup region. The results on peptide-membrane interactions are discussed in terms of possible uptake mechanisms.

Animals↗

PubMed2006

A polarized-light spectroscopy study of interactions of a hairpin polyamide with DNA.

We here study the interactions of a polyamide with large DNA, and compare to those of minor groove binder distamycin (DST), including high ligand/DNA binding ratios. Specific as well as nonspecific binding is probed using polarized-light spectroscopy combined with singular value decomposition analysis. Circular and linear dichroism data confirm binding geometries consistent with minor groove binding for both of the ligands. Interestingly, at high and intermediate ligand/DNA ratios the polyamide exhibits no significant sequence discrimination between mixed-sequence (calf thymus) and AT DNA as compared to DST. Each ligand is concluded to exhibit two different binding modes depending upon ligand/DNA ratio and nucleo-base sequence. At high binding ratios, distinct differences between the ligands are observed: circular dichroism spectra exciton effects provide evidence of bimolecular interactions of the polyamide when bound to AT-DNA, whereas no effects are seen with DST or mixed-sequence DNA. Also linear dichroism indicates that a change in binding geometry occurs at high polyamide/AT ratios, and that the effect occurs only with polyamide in contrast to DST. Since the effect is insignificant with DST, or with calf thymus DNA, it is concluded that it relates to the sizes of the ligands and the minor grooves, becoming critical in the limit of crowding.

Animals↗

PubMed2006

A biohybrid dynamic random access memory.

We report that GABA(A) receptors in a patch-clamped biological cell form a short-term memory circuit when integrated with a scanning-probe microfluidic device. Laminar patterns of receptor activators (agonists) provided by the microfluidic device define and periodically update the data input which is read and stored by the receptors as state distributions (based on intrinsic multistate kinetics). The memory is discharged over time and lasts for seconds to minutes depending on the input function. The function of the memory can be represented by an equivalent electronic circuit with striking similarity in function to a dynamic random access memory (DRAM) used in electronic computers. Multiplexed biohybrid memories may form the basis of large-scale integrated biocomputational/sensor devices with the curious ability to use chemical signals including odorants, neurotransmitters, chemical and biological warfare agents, and many more as input signals.

Animals↗

PubMed2006

Comparing mono- and divalent DNA groove binding cyanine dyes -- binding geometries, dissociation rates, and fluorescence properties.

The unsymmetrical cyanine dyes BOXTO-PRO and BOXTO-MEE were derived from the DNA groove binder BOXTO, by adding a positively charged or a non-ionic hydrophilic tail to BOXTO, respectively. The main objective was to obtain more efficient DNA probes, for instance in electrophoresis and microscopy, by slowing down the dissociation of BOXTO from DNA. The interactions with mixed sequence DNA was studied with fluorescence and absorbance spectroscopy, stopped-flow dissociation and gel electrophoresis. Both the derivatives are groove bound as BOXTO, and have similar fluorescence properties when bound to mixed sequence DNA in free solution. BOXTO-PRO exhibits a slower dissociation than BOXTO from DNA, whereas the dissociation rate for BOXTO-MEE is faster and, unexpectedly independent of the ionic strength. During gel electrophoresis both BOXTO-PRO and BOXTO-MEE exhibit a faster dissociation rate than BOXTO. Still, BOXTO-PRO seems to be a good alternative as DNA probe, especially for applications in free solution where the dissociation is slower than for the corresponding intercalator TOPRO-1.

Binding Sites↗

PubMed2005

Monitoring the DNA binding kinetics of a binuclear ruthenium complex by energy transfer: evidence for slow shuffling.

The semirigid binuclear ruthenium complex Delta,Delta-[mu-(11,11'-bidppz)(phen)(4)Ru(2)](4+) has been shown to rearrange slowly from an initial groove-bound nonluminescent state to a final intercalated emissive state by threading one of its bulky Ru(phen)(2) moieties through the DNA base stack. When this complex binds to poly[d(A-T)(2)], a further increase in emission from the complex is observed after completion of the intercalation, assigned to reorganization of the intercalated complex. We here report a study of the threading process in poly[d(A-T)(2)], in which the minor groove binding dye DAPI is used as an energy transfer probe molecule to assess the distribution of ruthenium complex during and also after the actual threading phase. The emission from DAPI is found to change with the same rate as the emission from the ruthenium complex, and furthermore, DAPI does not disturb the binding kinetics of the latter, justifying it as a good probe of both the threading and the reorganization processes. We conclude from the change in the emission from both DAPI and the ruthenium complex with time that DAPI-ruthenium interactions are most pronounced during the process of threading of the complex, suggesting that the complexes are initially threaded slightly anticooperatively and thereafter redistribute along the DNA to reach their thermodynamically most favorable distribution. The final distribution is characterized by a small but significant binding cooperativity, probably as a result of hydrophobic interactions between the complex ions despite their tetravalent positive charges. The mechanism of "shuffling" the complex along the DNA chain is discussed, i.e., whether the ruthenium complex remains threaded (requiring sequential base-pair openings) or if unthreading followed by lateral diffusion within the ionic atmosphere of the DNA and rethreading occurs.

DNA↗

PubMed2005

Enantioselective luminescence quenching of DNA light-switch [Ru(phen)2dppz]2+ by electron transfer to structural homologue [Ru(phendione)2dppz]2+.

The quenching of the luminescence of [Ru(phen)(2)dppz](2+) by structural homologue [Ru(phendione)(2)dppz](2+), when both complexes are bound to DNA, has been studied for all four combinations of Delta and Lambda enantiomers. Flow linear dichroism spectroscopy (LD) indicates similar binding geometries for all the four compounds, with the dppz ligand fully intercalated between the DNA base pairs. A difference in the LD spectrum observed for the lowest-energy MLCT transition suggests that a transition, potentially related to the final localization of the excited electron to the dppz ligand in [Ru(phen)(2)dppz](2+), is overlaid by an orthogonally polarized transition in [Ru(phendione)(2)dppz](2+). This would be consistent with a low-lying LUMO of the phendione moiety of [Ru(phendione)(2)dppz](2+) that can accept the excited electron from [Ru(phen)(2)dppz](2+), thereby quenching the emission of the latter. The lifetime of excited Delta-[Ru(phen)(2)dppz](2+) is decreased moderately, from 664 to 427 ns, when bound simultaneously with the phendione complex to DNA. The 108 ns lifetime of opposite enantiomer, Lambda-[Ru(phen)(2)dppz](2+), is only shortened to 94 ns. These results are consistent with an average rate constant for electron transfer of approximately 1.10(6) s(-1) between the phenanthroline- and phendione-ruthenium complexes. At binding ratios close to saturation of DNA, the total emission of the two enantiomers is lowered equally much, but for the Lambda enantiomer, this is not paralleled by a decrease in luminescence lifetime. A binding isotherm simulation based on a generalized McGhee-von Hippel approach shows that the Delta enantiomer binds approximately 3 times stronger to DNA both for [Ru(phendione)(2)dppz](2+) and [Ru(phen)(2)dppz](2+). This explains the similar decrease in total emission, without the parallel decrease in lifetime for the Lambda enantiomer. The simulation also does not indicate any significant binding cooperativity, in contrast to the case when Delta-[Rh(phi)(2)bipy](3+) is used as quencher. The very slow electron transfer from [Ru(phen)(2)dppz](2+) to [Ru(phendione)(2)dppz](2+), compared to the case when [Rh(phi)(2)phen](3+) is the acceptor, can be explained by a much smaller driving free-energy difference.

DNA↗

PubMed2005

Fluorescent properties of DNA base analogue tC upon incorporation into DNA--negligible influence of neighbouring bases on fluorescence quantum yield.

The quantum yield of the fluorescent tricyclic cytosine analogue, 1,3-diaza-2-oxophenothiazine, tC, is high and virtually unaffected by incorporation into both single- and double-stranded DNA irrespective of neighbouring bases (0.17-0.24 and 0.16-0.21, respectively) and the corresponding fluorescence decay curves are all mono-exponential, properties that are unmatched by any base analogue so far. The fluorescence lifetimes increase when going from tC free in solution (3.2 ns) to single- and double-stranded DNA (on average 5.7 and 6.3 ns, respectively). The mono-exponential decays further support previous NMR results where it was found that tC has a well-defined position and geometry within the DNA helix. Furthermore, we find that the oxidation potential of tC is 0.4 V lower than for deoxyguanosine, the natural base with the lowest oxidation potential. This suggests that tC may be of interest in charge transfer studies in DNA as an electron hole acceptor. We also present a novel synthetic route to the phosphoramidite form of tC. The results presented here together with previous work show that tC is a very good C-analogue that induces minimal perturbation to the native structure of DNA. This makes tC unique as a fluorescent base analogue and is thus highly interesting in a range of applications for studying e.g. structure, dynamics and kinetics in nucleic acid systems.

DNA↗

PubMed2005

Mechanism of DNA threading intercalation of binuclear Ru complexes: uni- or bimolecular pathways depending on ligand structure and binding density.

In the long succession of small transition-metal compounds interacting reversibly with DNA, semirigid binuclear ruthenium complexes stand out by displaying exceptionally slow binding kinetics. To reach the final intercalated state, one of the bulky metal centers has to be threaded through the base stack, leading to a high level of structural discrimination. This makes the idea of utilizing binuclear complexes interesting in applications involving DNA sequence or conformation recognition. The finding that threading intercalation of the two structural analogues, Lambda,Lambda-[mu-(11,11'-bidppz)X4Ru2]4+, X = 2,2'-bipyridine (Lambda,Lambda-B4) and X = 1,10'-phenanthroline (Lambda,Lambda-P4), into poly(dA-dT)2 can be described by surprisingly simple rate laws encouraged more extensive studies and analysis of these two systems. Kinetic measurements at different [basepair]/[complex] ratios show that Lambda,Lambda-B4 intercalates via a pseudo-first-order mechanism independent of binding density, whereas Lambda,Lambda-P4 displays a gradual transition from apparent first- to second-order kinetics when decreasing the [basepair]/[complex] mixing ratio. By employing the probabilistic method of McGhee and von Hippel, a rate law based on a supposed mechanism has been globally fitted and numerically integrated to describe threading of Lambda,Lambda-P4. In contrast to Lambda,Lambda-B4, the first-order mechanism of this analogue appears to require a long stretch of nonthreaded DNA. The results show that ancillary ligand structures indeed affect the mechanism of DNA threading, demonstrating the potential use of semirigid binuclear ruthenium complexes to target DNA.

DNA↗

PubMed2004

Membrane destabilizing properties of cell-penetrating peptides.

Although cell-penetrating peptides (CPPs), also denoted protein transduction domains (PTDs), have been widely used for intracellular delivery of large and hydrophilic molecules, the mechanism of uptake is still poorly understood. In a recent live cell study of the uptake of penetratin and tryptophan-containing analogues of Tat(48-60) and oligoarginine, denoted TatP59W, TatLysP59W and R(7)W, respectively, it was found that both endocytotic and non-endocytotic uptake pathways are involved [Thoren et al., Biochem. Biophys. Res. Commun. 307 (2003) 100-107]. Non-endocytotic uptake was only observed for the arginine-rich peptides TatP59W and R(7)W. In this paper, the interactions of penetratin, R(7)W, TatP59W and TatLysP59W with phospholipid vesicles are compared in the search for an understanding of the mechanisms for cellular uptake. While R(7)W, TatP59W and TatLysP59W are found to promote vesicle fusion, indicated by mixing of membrane components, penetratin merely induces vesicle aggregation. Studies of the leakage from dye-loaded vesicles indicate that none of the peptides forms membrane pores and that vesicle fusion is not accompanied by leakage of the aqueous contents of the vesicles. These observations are important for a proper interpretation of future experiments on the interactions of these peptides with model membranes. We suggest that the discovered variations in propensity to destabilize phospholipid bilayers between the peptides investigated, in some cases sufficient to induce fusion, may be related to their different cellular uptake properties.

Arginine↗

PubMed2004

Effects of methyl substitution on radiative and solvent quenching rate constants of [Ru(phen)2dppz]2+ in polyol solvents and bound to DNA.

Methyl substituents on the distant benzene ring of the dppz ligand in the "light switch" complex [Ru(phen)(2)dppz](2+) have profound effects on the photophysics of the complexes in water as well as in the polyol solvents ethylene glycol, glycerol, and 1,2- and 1,3-propanediol. Whereas 11,12-dimethyl substitution decreases the rate of quenching by diminishing hydrogen bonding by solvent, the 10-methyl substituent in addition also decreases both the radiative and the nonradiative rate constant for decay to the ground state of the non-hydrogen-bonded excited state species. For both the 10-methyl and the 11,12-dimethyl derivatives, the effect of methyl substitution on the equilibrium of solvent hydrogen bonding to the excited state is due to changes in the entropy terms, rather than in the enthalpy, indicating that the effect is a steric perturbation of the solvent cage around the molecule. When intercalated into DNA, the effects of methyl substitution is smaller than those in polyol solvent or water, suggesting that the water molecules that quench the excited state by hydrogen bonding to the phenazine aza nitrogens mainly access them from the same groove as in which the Ru(II) ion resides. Since the Delta-enantiomer of [Ru(phen)(2)10-methyl-dppz](2+) has an absolute quantum yield of up to 0.23 when bound to DNA, a value 7000 times higher than in pure water solution, it is promising as a new luminescent DNA probe.

Absorption↗

PubMed2004

Vesicle size-dependent translocation of penetratin analogs across lipid membranes.

The recent discoveries of serious artifacts associated with the use of cell fixation in studies of the cellular uptake of cell-penetrating peptides (CPPs) have prompted a reevaluation of the current understanding of peptide-mediated cellular delivery. Following a report on the differential cellular uptake of a number of penetratin analogs in unfixed cells, we here investigate their membrane translocation abilities in large and giant unilamellar vesicles (LUVs and GUVs, respectively). Surprisingly, in contrast to the behavior in living cells, all peptides readily entered the giant vesicles (>1 microm) as proved by confocal microscopy, while none of them could cross the membranes of LUVs (100 nm). For determination of the location of the peptides in the LUVs, a new concept was introduced, based on sensitive resonance energy transfer (RET) measurements of the enhanced fluorescence of acceptor fluorophores present solely in the inner leaflet. An easily adopted method to prepare such asymmetrically labeled liposomes is described. The membrane insertion depths of the tryptophan moieties of the peptides were determined by use of brominated lipids and found to be very similar for all of the peptides studied. We also demonstrate that infrared spectroscopy on the lipid carbonyl stretch vibration peak is a convenient technique to determine phospholipid concentration.

Biological Transport↗

PubMed2004

DNA adopts normal B-form upon incorporation of highly fluorescent DNA base analogue tC: NMR structure and UV-Vis spectroscopy characterization.

The influence of the highly fluorescent tricyclic cytosine base analogue (tC) on duplex DNA conformation is investigated. The duplex properties are characterized by absorbance and circular dichroism (CD) for all combinations of neighbouring bases to tC, and an NMR structure is determined for one tC-containing sequence. For the oligonucleotides with one tC incorporated instead of cytosine, the melting temperature is increased on average by 2.7 degrees C above that for the unmodified ones. CD spectra are practically identical for modified and unmodified sequences, indicating an unperturbed B-DNA conformation. The NMR structure determination of the self-complementary sequence 5'-CTC(tC)ACGTGGAG shows a DNA conformation consistent with B-form for the whole duplex. The root-mean-square distance for the nucleotides of the eight central base pairs between the 10 structures with lowest CYANA target functions and a mean structure is 0.45 +/- 0.17 A. The NMR data confirm correct base pairing for tC by the observation of both intrastrand and interstrand imino proton NOEs. Altogether, this suggests that tC works well as a cytosine analogue, i.e. it is situated in the base stack, forming hydrogen bonds with G in the complementary strand, without distorting the DNA backbone conformation. This first example of an artificial, highly fluorescent DNA base that does not perturb the DNA conformation could have valuable applications for the study of the structure and dynamics of nucleic acid systems.

Circular Dichroism↗

PubMed2004

Vesicle membrane interactions of penetratin analogues.

Reports on serious artifacts associated with the use of cell fixation in studies of the cellular uptake of cell-penetrating peptides, also denoted protein transduction domains, have demonstrated the need for a reevaluation of the current understanding of peptide-mediated cellular delivery of large, hydrophilic molecules. In a recent study on the internalization in unfixed cells of penetratin and its analogues in which tryptophans are substituted for phenylalanines (Pen2W2F), lysines for arginines (PenArg), and arginines for lysines (PenLys), we revealed large dissimilarities in cell interactions among the peptides [Thorén et al. (2003) Biochem. Biophys. Res. Commun. 307, 100-107]. We here investigated possible correlations with their respective affinities for the lipid membranes of large unilamellar vesicles. The variations found in membrane affinity correlated qualitatively with differences in hydrophobicity among the peptides but were by far too small to account for the striking differences in cell membrane binding. Interestingly, we found that the inclusion of a small fraction of lipids conjugated to poly(ethylene glycol) (PEG) in the vesicles both stabilized the vesicle dispersion against peptide-induced aggregation and, furthermore, enhanced the binding of the peptides to the membrane. By use of PEG-conjugated lipids, it could be shown that vesicle aggregation drives an alpha-helix to beta-sheet conformational transition for these peptides. A similar transition was discovered at submicellar concentrations of sodium dodecyl sulfate in aqueous solution for all peptides except PenLys. Finally, significant changes of the contributions to CD spectra from aromatic residues due to their insertion into the membrane were observed.

Amino Acid Sequence↗

PubMed2004

Syntheses and DNA-binding studies of a series of unsymmetrical cyanine dyes: structural influence on the degree of minor groove binding to natural DNA.

Twelve crescent-shaped unsymmetrical dyes have been synthesized and their interactions with DNA have been investigated by spectroscopic methods. A new facile synthetic route to this type of cyanine dyes has been developed, involving the preparation of 6-substituted 2-thiomethyl-benzothiazoles in good yields. The new dyes are analogues to the minor groove binding unsymmetrical cyanine dye, BEBO, recently reported by us. In this dye, the structure of the known intercalating cyanine dye BO was extended with a 6-methylbenzothiazole substituent. Herein we further investigate the role of the extending benzazole heterocycle, as well as of the pyridine or quinoline moiety of the cyanine chromophore, for the binding mode of these crescent-shaped dyes to calf thymus DNA. Flow LD and CD studies of the 12 dyes show that the extent of minor groove binding to mixed sequence DNA varies significantly between the dyes. We find that hydrophobicity and size are the crucial parameters for recognition of the minor groove. The relatively high fluorescence quantum yield of many of these cyanines bound to DNA, combined with their absorption at long wavelengths, may render them useful in biological applications. In particular, two of the benzoxazole containing dyes BOXTO and 2-BOXTO show a high degree of minor groove binding and quantum yields of 0.52 and 0.32, respectively, when bound to DNA.

Binding Sites↗

PubMed2004

Ambivalent intercalators for DNA: L-shaped platinum(II) complexes.

Novel platinum(II) square planar coordination complexes, in which two heteroaromatic ligands are held by the metal in an unusual L-shaped geometry orthogonal to each other, have been synthesized, and their interaction with DNA was investigated with absorption and linear dichroism spectroscopy. As a rule, the ligand that is coplanar with the coordination square of Pt is found to be oriented perpendicular relative to the DNA helix axis when bound, suggestive of its intercalation between the base pairs of DNA. However, when this coplanar ligand is replaced by two pyridines, the opposite ligand, orthogonal to the coordination square, is instead preferentially intercalated. This behavior shows that these new complexes do indeed show some properties of true ambintercalators, i.e., compounds that can bind by intercalation of either of two distinct aromatic moieties.

DNA↗

PubMed2004

Membrane binding and translocation of cell-penetrating peptides.

Cell-penetrating peptides (CPPs) have been extensively studied during the past decade, because of their ability to promote the cellular uptake of various cargo molecules, e.g., oligonucleotides and proteins. In a recent study of the uptake of several analogues of penetratin, Tat(48-60) and oligoarginine in live (unfixed) cells [Thorén et al. (2003) Biochem. Biophys. Res. Commun. 307, 100-107], it was found that both endocytotic and nonendocytotic uptake pathways are involved in the internalization of these CPPs. In the present study, the membrane interactions of some of these novel peptides, all containing a tryptophan residue to facilitate spectroscopic studies, are investigated. The peptides exhibit a strong affinity for large unilamellar vesicles (LUVs) containing zwitterionic and anionic lipids, with binding constants decreasing in the order penetratin > R(7)W > TatP59W > TatLysP59W. Quenching studies using the aqueous quencher acrylamide and brominated lipids indicate that the tryptophan residues of the peptides are buried to a similar extent into the membrane, with an average insertion depth of approximately 10-11 A from the bilayer center. The membrane topology of the peptides was investigated using an assay based on resonance energy transfer between tryptophan and a fluorescently labeled lysophospholipid, lysoMC, distributed asymmetrically in the membranes of LUVs. By determination of the energy transfer efficiency when peptide was added to vesicles with lysoMC present exclusively in the inner leaflet, it was shown that none of the peptides investigated is able to translocate across the lipid membranes of LUVs. By contrast, confocal laser scanning microscopy studies on carboxyfluorescein-labeled peptides showed that all of the peptides rapidly traverse the membranes of giant unilamellar vesicles (GUVs). The choice of model system is thus crucial for the conclusions about the ability of CPPs to translocate across lipid membranes. Under the conditions used in the present study, peptide-lipid interactions alone cannot explain the different cellular uptake characteristics exhibited by these peptides.

Acrylamide↗