The coupling of bacterial surface (S)-layer proteins to lipid membranes is studied in molecular detail for proteins from Bacillus sphaericus CCM2177 and B. coagulans E38-66 recrystallized at dipalmitoylphosphatidylethanolamine (DPPE) monolayers on aqueous buffer. A comparison of the monolayer structure before and after protein recrystallization shows minimal reorganization of the lipid chains. By contrast, the lipid headgroups show major rearrangements. For the B. sphaericus CCM2177 protein underneath DPPE monolayers, x-ray reflectivity data suggest that amino acid side chains intercalate the lipid headgroups at least to the phosphate moieties, and probably further beyond. The number of electrons in the headgroup region increases by more than four per lipid. Analysis of the changes of the deduced electron density profiles in terms of a molecular interpretation shows that the phosphatidylethanolamine headgroups must reorient toward the surface normal to accommodate such changes. In terms of the protein structure (which is as yet unknown in three dimensions), the electron density profile reveals a thickness lz approximately 90 A of the recrystallized S-layer and shows water-filled cavities near its center. The protein volume fraction reaches maxima of >60% in two horizontal sections of the S-layer, close to the lipid monolayer and close to the free subphase. In between it drops to approximately 20%. Four S-layer protein monomers are located within the unit cell of a square lattice with a spacing of approximately 131 A.

We have designed and synthesized original cationic lipids for modulated release of DNA from cationic lipid/DNA complexes. Our rationale was that modulated degradation of the lipids during or after penetration into the cell could improve the trafficking of DNA to the nucleus resulting in increased transgene expression. The new reduction-sensitive lipopolyamines (RSL) harbor a disulfide bridge within different positions in the backbone of the lipids as biosensitive function. A useful synthetic method was developed to obtain, with very good yields and reproducibility, unsymmetrical disulfide-bridged molecules, starting from symmetrical disulfides and thiols. The new lipopolyamines are good candidates as carriers of therapeutic genes for in vivo gene delivery. To optimize the transfection efficiency in these novel series, we have carried out structure-activity relationship studies by placing the disulfide bridge at different positions in the backbone of the cationic lipid and by systematic variation of lipid chain length. Results indicate that the transfection level can be modulated as a function of the location of the disulfide bridge in the molecule. We suggest that an early release of DNA during or after penetration into the cell, probably promoted by reduction of a disulfide bridge placed between the polyamine and the lipid, implies a total loss of transfection efficiency. On the other hand, proper modulation of DNA release by inserting the disulfide bridge between one lipid chain and the rest of the molecule brings about increased transfection efficiency as compared to previously described nondegradable lipopolyamine analogues. Finally, preliminary physicochemical characterization of the complexes demonstrates that DNA release from complexes can be modulated as a function of the surrounding reducing conditions of the complexes and of the localization of the disulfide bridge within the lipopolyamine. Our results suggest that RSL is a promising new approach for gene delivery.

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Our results represent an important step towards the design of multimodular BGTC-based systems for improved in vivo gene transfection.

One of the main challenges of gene therapy remains the increase of gene delivery into eukaryotic cells. We tested whether intracellular DNA release, an essential step for gene transfer, could be facilitated by using reducible cationic DNA-delivery vectors. For this purpose, plasmid DNA was complexed with cationic lipids bearing a disulphide bond. This reduction-sensitive linker is expected to be reduced and cleaved in the reducing milieu of the cytoplasm, thus potentially improving DNA release and consequently transfection. The DNA--disulphide-lipid complexation was monitored by ethidium bromide exclusion, and the size of complexes was determined by dynamic light scattering. It was found that the reduction kinetics of disulphide groups in DNA--lipid complexes depended on the position of the disulphide linker within the lipid molecule. Furthermore, the internal structure of DNA--lipid particles was examined by small-angle X-ray scattering before and after lipid reduction. DNA release from lipid complexes was observed after the reduction of disulphide bonds of several lipids. Cell-transfection experiments suggested that complexes formed with selected reducible lipids resulted in up to 1000-fold higher reporter-gene activity, when compared with their analogues without disulphide bonds. In conclusion, reduction-sensitive groups introduced into cationic lipid backbones potentially allow enhanced DNA release from DNA--lipid complexes after intracellular reduction and represent a tool for improved vectorization.

The recrystallization of the S-layer protein from Bacillus coagulans E38-66 at different lipid surface monolayer films has been observed to depend on (i) the nature of the lipid headgroup, (ii) the phase state of the surface monolayer, (iii) the ionic content, and (iv) the pH of the subphase. S-layer lattices formed at such interfaces were studied by electron microscopy and their orientation with respect to the lipid films was determined from the handedness of the base vectors defining the oblique crystal lattice. Recrystallization of S-layer lattices that cover the entire sample area was generally observed at lipids with zwitterionic headgroups in the presence of Ca2+ if the lipid chains possessed a high degree of order, i.e., if the lipid film was in the liquid condensed phase. Under such conditions, the S-layer was found to be attached to the lipid with its net negatively charged inner face. In contrast, the S-layer protein recrystallized poorly under most lipids with negatively charged headgroups and under lipids with unsaturated chains. At monolayers of cationic lipids, reconstituted S-layers were observed in which the protein was attached to the lipid with its outer face.

Structural details of the coupling of bacterial surface (S)-layers to the phospholipid, dipalmitoylphosphatidylethanolamine (DPPE), have been characterized using X-ray and neutron reflectometry. We studied the binding and recrystallization of S-protein isolated from B. sphaericus CCM2177 at DPPE monolayers on aqueous surfaces. Particular emphasis has been put on investigations of the lipid/protein interface in a joint refinement of X-ray and neutron data which reveals alterations of the molecular-level organization of the lipid headgroups upon protein binding and recrystallization:  Peptide material interpenetrates the phospholipid headgroups almost in its entire depth but does not affect the hydrophobic lipid acyl chains. Consistent with FTIR results, we find that the headgroup hydration is reduced by ∼40% upon peptide interpenetration. On average, the equivalent of ∼65 electrons associated with the peptide, i.e., less than one peptide side group, interacts directly with one DPPE headgroup within the surface film. This suggests that the protein attaches to specific molecular moieties within the lipid monolayer which may form a lateral pattern within the film area that reflects the properties of the monomolecular protein crystal sheet.

Supported lipid bilayers on planar silicon substrates have been formed using crystalline bacterial cell surface (S-layer) protein as support onto which DMPC (pure or mixture with 30 mol % cholesterol) or DPPC bilayers were deposited. Lateral diffusion of fluorescence lipid probes in these layers have been investigated with fluorescence recovery after photobleaching technique (FRAP). For comparison, hybrid lipid bilayers (lipid monolayer on alkylsilanes) and lipid bilayers on dextran composed of the same lipids as for S-layer-supported systems were studied. The mobility of lipids was highest in the S-layer-supported bilayers. No significant difference in mobility was observed for supports of the two S-layer proteins from Bacillus coagulans E38-66 or Bacillus sphaericus CCM2177. DMPC/cholesterol-layers revealed mostly a homogeneous structure, whereas in planar DPPC layers defects could be observed. In S-layer-supported DPPC bilayers, clear cracks could be seen below T m whereas above T m inhomogeneous round structures were formed. In another set of experiments the supported bilayers have been covered by S-layer proteins using three different techniques for protein recrystallization (trough, vertical, and horizontal). The recrystallization of S-layers was visualized in large scale by electron microscopy (EM) and more specific on the different substrates by atomic force microscopy (AFM). The S-layer cover induced an enhanced mobility of the probe in the lipid layer. Furthermore it was noticed that the S-layer lattice cover could prevent the formation of cracks and other inhomogenities in the bilayers.