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-<i>sn</i>-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 (<i>LC</i>) domains even at Π values where the monolayer is supposedly in the liquid-expanded (<i>LE</i>) 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 <i>LC</i>-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 <i>LE</i> to <i>LC</i> state is smeared out or is shifted to higher surface pressure. Considerable changes in the shapes of <i>LC</i>-domains were observed after peptide binding. Growth of the <i>LC</i>-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

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-<i>sn</i>-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