Membrane binding properties of the C-terminal segment of retinol dehydrogenase 8.

Light absorption by rhodopsin leads to the release of all-trans retinal (ATRal) in the lipid phase of photoreceptor disc membranes. Retinol dehydrogenase 8 (RDH8) then reduces ATRal into all-trans retinol, which is the first step of the visual cycle. The membrane binding of RDH8 has been postulated to be mediated by one or more palmitoylated cysteines located in its C-terminus. Different peptide variants of the C-terminus of RDH8 were thus used to obtain information on the mechanism of membrane binding of this enzyme. Steady-state and time-resolved fluorescence measurements were performed using short and long C-terminal segments of bovine RDH8, comprising one or two tryptophan residues. The data demonstrate that the amphipathic alpha helical structure of the first portion of the C-terminus of RDH8 strongly contributes to its membrane binding, which is also favored by palmitoylation of at least one of the cysteines located in the last portion of the C-terminus.

Interaction of Short Pentavalent Cationic Peptides with Negatively Charged DPPG Monolayers and Bilayers: Influence of Peptide Modifications on Binding.

The binding of oligopeptides with the structure (RX)4R and (KXX)4K, with X being the amino acid G or A, to lipid monolayers and bilayers of dipalmitoyl-phosphatidylglycerol (DPPG) was studied and compared to the binding effects of peptides with the structure (KX)4K. The monolayer adsorption experiments again showed the superposition of condensation effects due to charge compensation and insertion of amino acid side chains leading to expansion of the monolayer. The latter effect was enhanced when glycine was replaced by alanine. The thermotropic phase behavior of dipalmitoyl-phosphatidylglycerol (DPPG) bilayer membranes and their mixtures with short cationic model peptides was investigated by differential scanning calorimetry and infrared spectroscopy. Increasing the charge distance of the lysine residues in the series (K)5, (KG)4K, and (KGG)4K results in an upshift of the main phase transition of DPPG up to 5 K, as predicted for pure electrostatic binding. All peptides exhibit only unordered structures in bulk solution as well as when bound to DPPG bilayers. (KGG)4K additionally shows a high propensity of turn structures due to its flexibility. The exchange of glycine by alanine in (KAA)4K leads only to a marginal increase in Tm, in contrast to the binding of (KA)4K where the formation of intervesicular antiparallel ß-sheets occurs, leading to a much more pronounced stabilization of the gel phase. This shows that the sequence and flexibility of the oligopeptides has an important influence on the formation of secondary structures bound to the bilayers. Binding of (RX)4R peptides to DPPG bilayers has almost no influence on the lipid phase transition in bilayers. Here, condensation and insertion effects almost compensate, as the results of monolayer experiments show. This is due to the higher propensity of arginine side chains to insert into the lipid headgroup region.

Cospreading of Anionic Phospholipids with Peptides of the Structure (KX)4K at the Air-Water Interface: Influence of Lipid Headgroup Structure and Hydrophobicity of the Peptide on Monolayer Behavior.

Mixtures of anionic phospholipids (PG, PA, PS, and CL) with cationic peptides were cospread from a common organic solvent at the air-water interface. The compression of the mixed film was combined with epifluorescence microscopy or infrared reflection adsorption spectroscopy (IRRAS) to gain information on the interactions of the peptide with the different lipids. To evaluate the influence of the amino acid X of peptides with the sequence (KX)4K on the binding, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) was mixed with different peptides with increasing hydrophobicity of the uncharged amino acid X. The monolayer isotherms of DPPG/(KX)4K mixtures show an increased area for the lift-off due to incorporation of the peptide into the liquid-expanded (LE) state of the lipid. The surface pressure for the transition from LE to the liquid-condensed (LC) state is slightly increased for peptides with amino acids X with moderate hydrophobicity. For the most hydrophobic peptide (KL)4K two plateaus are seen at a charge ratio PG to K of 5:1, and a strongly increased transition pressure is observed for a charge ratio of 1:1. Epifluorescence microscopy images and infrared spectroscopy show that the lower plateau corresponds to the LE-LC phase transition of the lipid. The upper plateau is connected with a squeeze-out of the peptide into the subphase. To test the influence of the lipid headgroup structure on peptide binding (KL)4K was cospread with different anionic phospholipids. The shift of the isotherm to larger areas for lift-off and to higher surface pressure for the LE-LC phase transition was observed for all tested anionic lipids. Epifluorescence microscopy reveals the formation of LC domains with extended filaments indicating a decrease in line tension due to accumulation of the peptides at the LC-domain boundaries. This effect depends on the size of the headgroup of the anionic phospholipid.

Interaction of DNA with Cationic Lipid Mixtures-Investigation at Langmuir Lipid Monolayers.

Four different binary lipid mixtures composed of a cationic lipid and the zwitterionic colipids DOPE or DPPC, which show different DNA transfer activities in cell culture models, were investigated at the soft air/water interface to identify transfection efficiency determining characteristics. Langmuir films are useful models to investigate the interaction between DNA and lipid mixtures in a two-dimensional model system by using different surface sensitive techniques, namely, epifluorescence microscopy and infrared reflection-absorption spectroscopy. Especially, the effect of adsorbed DNA on the properties of the lipid mixtures has been examined. Distinct differences between the lipid composites were found which are caused by the different colipids of the mixtures. DOPE containing lipid mixtures form fluid monolayers with a uniform distribution of the fluorescent probe in the presence and absence of DNA at physiologically relevant surface pressures. Only at high nonphysiological pressures, the lipid monolayer collapses and phase separation was observed if DNA was present in the subphase. In contrast, DPPC containing lipid mixtures show domains in the liquid condensed phase state in the presence and absence of DNA in the subphase. The adsorption of DNA at the positively charged mixed lipid monolayer induces phase separation which is expressed in the morphology and the point of appearance of these domains.

Binding of cationic model peptides (KX)4K to anionic lipid bilayers: Lipid headgroup size influences secondary structure of bound peptides.

Differential Scanning Calorimetry (DSC) and Fourier transformed Infrared (FT-IR) spectroscopy were used to test the influence of acyl chain length, acyl chain saturation, and chemical structure of anionic phospholipids on the interaction with cationic model peptides (KX)4K, with amino acid X=A, Abu, and L. The lipids used were phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA), and cardiolipin (CL). DSC was used to monitor the phase transition of lipid vesicles before and after peptide binding. The electrostatic attraction is the main driving force for binding. The hydrophobicity of the amino acid X influences the binding strength as well as the secondary structure of the bound peptide. Binding of peptides leads to an upshift of the lipid phase transition. Lipids with smaller headgroups show a larger upshift of the main phase transition temperature. Data from FT-IR spectroscopy show in addition that the stability of the bound ß-sheets of (KX)4K depends on the hydrophobicity of the uncharged amino acid X and on the size of the lipid headgroup. For lipids with large anionic headgroups, such as PS, the antiparallel ß-sheet of (KAbu)4K bound to gel phase bilayers is converted to an unordered structure upon heating through the lipid phase transition. Reducing the size of the headgroup, as in PG, increases the stability of the bound peptide ß-sheets. For the smallest headgroups, present in PA and CL, stably bound ß-sheets are observed even above the lipid phase transition.

Binding of the Cationic Peptide (KL)4K to Lipid Monolayers at the Air-Water Interface: Effect of Lipid Headgroup Charge, Acyl Chain Length, and Acyl Chain Saturation.

The binding of the cationic peptide (KL)4K to monolayers of different anionic lipids was determined by adsorption experiments. The chemical structure of the anionic phospholipids was changed in different ways. First, the hydrophobic region of phosphatidylglycerols was altered by elongation of the acyl chain length. Second, an unsaturated chain was introduced. Third, lipids with negatively charged headgroups of different chemical structure were compared. (KL)4K itself shows no surface activity and does not bind to monolayers of zwitterionic lipids. Analysis of (KL)4K binding to anionic lipid monolayers reveals a competition between two binding processes: (i) incorporation of the peptide into the acyl chain region (surface pressure increase) and (ii) electrostatic interaction screening the negative charges with reduction of charge repulsion (surface pressure decrease due to monolayer condensation). The lipid acyl chain length and the chemical structure of the headgroup have minor effects on the binding properties. However, a strong dependence on the phase state of the monolayer was observed. In the liquid-expanded (LE) phase, the fluid monolayer provides enough space, so that peptide insertion due to hydrophobic interactions dominates. For monolayers in the liquid-condensed (LC) phase, peptide binding followed by monolayer condensation is the main effect.

Binding of cationic peptides (KX)4K to DPPG bilayers. Increasing the hydrophobicity of the uncharged amino acid X drives formation of membrane bound ß-sheets: A DSC and FT-IR study.

The binding of cationic peptides of the sequence (KX)4K to lipid vesicles of negatively charged dipalmitoyl-phosphatidylglycerol (DPPG) was investigated by differential scanning calorimetry (DSC) and temperature dependent Fourier-transformed infrared (FT-IR) spectroscopy. The hydrophobicity of the uncharged amino acid X was changed from G (glycine) over A (alanine), Abu (α-aminobutyric acid), V (valine) to L (leucine). The binding of the peptides caused an increase of the phase transition temperature (Tm) of DPPG by up to 20°C. The shift depended on the charge ratio and on the hydrophobicity of the amino acid X. Unexpectedly, the upward shift of Tm increased with increasing hydrophobicity of X. FT-IR spectroscopy showed a shift of the CH2 stretching vibrations of DPPG to lower frequency, particularly for bilayers in the liquid-crystalline phase, indicating an ordering of the hydrocarbon chains when the peptides were bound. Changes in the lipid C=O vibrational band indicated a dehydration of the lipid headgroup region after peptide binding. (KG)4K was bound in an unordered structure at all temperatures. All other peptides formed intermolecular antiparallel ß-sheets, when bound to gel phase DPPG. However, for (KA)4K and (KAbu)4K, the ß-sheets converted into an unordered structure above Tm. In contrast, the ß-sheet structures of (KV)4K and (KL)4K remained stable even at 80°C when bound to the liquid-crystalline phase of DPPG. Strong aggregation of DPPG vesicles occurred after peptide binding. For the aggregates, we suggest a structure, where aggregated single ß-sheets are sandwiched between opposing DPPG bilayers with a dehydrated interfacial region.

Binding of Short Cationic Peptides (KX)4K to Negatively Charged DPPG Monolayers: Competition between Electrostatic and Hydrophobic Interactions.

The influence of the peptide sequence on the binding of short cationic peptides composed of five lysines alternating with uncharged amino acids within the series (KX)4K to negatively charged monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) was investigated by adsorption experiments in combination with epifluorescence microscopy. To evaluate the impact of electrostatic and hydrophobic contributions, different uncharged amino acids X with increasing hydrophobicity, where X = G (glycine), A (alanine), Abu (α-aminobutyric acid), V (valine), or L (leucine) were introduced into the peptide sequence to tune the peptide hydrophobicity. The adsorption kinetics of these peptides to a DPPG monolayer always showed two superimposed processes, one leading to an increase and another to a decrease of the surface pressure Π. Thus, the plots of the change in Π after peptide binding vs initial surface pressure of the monolayer showed an unusual behavior with maxima and negative changes in Π at high initial Π values. Epifluorescence microscopy confirmed that electrostatic binding of the peptides with a concomitant decrease in Π leads to a condensation of the lipid monolayer and the formation of liquid-condensed (LC) domains even at Π values where the monolayer is supposedly in the liquid-expanded (LE) state. An increase in hydrophobicity of the amino acid X was found to counteract the condensation and an increase in Π upon peptide binding is observed at low Π values, also concomitant with the formation of LC-domains. Compression of monolayers after peptide adsorption at low surface pressure for 4 h leads to a change of the isotherms compared to pure DPPG isotherms. The phase transition of DPPG from LE to LC state is smeared out or is shifted to higher surface pressure. Considerable changes in the shapes of LC-domains were observed after peptide binding. Growth of the LC-domains was hindered in most cases and regular domain patterns were formed. Binding of (KL)4K leads to a decrease in line tension and the formation of extended filaments protruding from initially circular domains.

Interactions of Pluronic block copolymers with lipid monolayers studied by epi-fluorescence microscopy and by adsorption experiments.

The interactions of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers, i.e. Pluronics F87, F88 and F127, with monolayers composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were investigated with different monolayer techniques. Surface pressure-area isotherms were recorded of co-spread Pluronic/lipid mixtures with different Pluronic content to determine the influence of the polymers on the monolayer phase transitions. The squeeze-out pressure of the polymers upon film compression was dependent on the PPO block length. The monolayer compression experiments were coupled with fluorescence microscopy to visualize the phase separation into polymer-rich and lipid-rich domains and to monitor morphological changes of the lipid domains in the monolayer. Extensive phase separation was observed in the coexistence region between liquid-expanded (LE) and liquid-condensed (LC) lipid phases, where pure polymer domains coexisting with round LE-domains containing polymer, and polymer-free LC-domains were seen. We also investigated the adsorption of Pluronics to a lipid monolayer after injecting a polymer solution underneath a pre-formed lipid monolayer by following the change in pressure at constant area. The results show that polymer adsorption is a superposition of two individual processes with different kinetics. Pluronics with a higher hydrophobicity and with a smaller molecular weight adsorb faster and the type and phase state of the lipid determines the surface pressure where no further Pluronic molecules adsorb to the interface. This critical surface pressure depends on the PPO block length, whereas the strength of the interaction with the lipids is determined by the relative PEO content. This indicates that also interactions between the PEO blocks and the lipid headgroup region are occurring. The interactions with the unsaturated lipid POPC in the liquid-expanded phase turn out to be stronger than for lipids in the liquid-condensed phase, where the polymers are excluded.