Two-dimensional crystallization of a bacterial surface protein on lipid vesicles under controlled conditions

Abstract

The solubilized surface protein of the Gram-negative bacterium Comamonas acidovorans was reconstituted on lipid vesicles by means of controlled dialysis. To this end, a multichamber dialysis apparatus was built which allows one to control the temperature and the dialysis rate, to apply various temperatures or buffer systems and sample conditions in a single experiment, and to monitor the turbidity of the sample by means of light scattering. The reconstitution conditions were optimized such that the surface protein formed two-dimensional crystals suitable for electron crystallography. The recrystallized surface protein arrays gave a resolution of approximately 1.3 nm in projection after correlation averaging of negatively stained preparations. The surface protein assembled into partially self-contained two-dimensional crystals which possess a strong shape-determining effect and formed cylinders and various cone-shaped vesicles. The development of the various vesicle forms is described in a model.

Are S-layers exoskeletons? The basic function of protein surface layers revisited

“…The lipid molecules are immobilised indirectly by non-specific association to the Slayer protein whereby the membranes become less fluid, less flexible, more stable and heatresistant, and presumably more resistant to hydrostatic pressure. The S-layer protein from Delftia acidovorans (formerly Comamonas acidovorans) tightly binds LPS and can be reconstituted on dimyristoyl phosphatidylcholine (DMPC) membranes via hydrophobic anchoring Paul et al, 1992). Fourier-transform infrared spectroscopy revealed an altered phase transition behavior of the lipid membrane ( Fig.…”

“…They are even capable of reshaping lipid vesicles made of DMPC upon forming selfcontained lattices ( Fig. 2; Paul et al, 1992). However, if the formation of self-contained lattices were a principle of shape determination, why then are other archaea not shaped accordingly, especially as 2-D lattices from p1 to p6 symmetries have the capacity to form selfcontained cylinders (Paul et al, 1992)?…”

“…2; Paul et al, 1992). However, if the formation of self-contained lattices were a principle of shape determination, why then are other archaea not shaped accordingly, especially as 2-D lattices from p1 to p6 symmetries have the capacity to form selfcontained cylinders (Paul et al, 1992)? The cells of Sulfolobales (Brock, 1981;Prüschenk et al, 1987), Desulfurococcales , Nanoarchaeum (Huber et al, 2002;Briegel, 2005), Archaeoglobus (Kessel et al, 1990) and of other archaea are rounded or irregularly shaped in thin-sectioned preparations.…”

Characterization of a porin from the outer membrane of Vibrio anguillarum

“…The concentration of the MOMP was determined by the bicinchoninic acid assay (46). The concentration of the detergent was assessed by measuring the diameters of 10-l droplets deposited on a hydrophobic surface (39). For conductance measurements, a portion of the preparation was used for anion-exchange chromatography with a MonoQ column (Pharmacia) equilibrated with 10 mM methylamine buffer, pH 8.5, plus 0.5% octyl-POE.…”

“…The solubilized MOMP was two-dimensionally crystallized in reconstituted membranes by detergent removal in controlled dialysis. In order to optimize the crystallization conditions, several experiments were performed in a multichamber dialysis apparatus, which allows up to seven samples to be treated independently in a single experiment as previously described (39). In all the samples, the protein concentration was adjusted to 1 mg/ml and the initial octyl-POE content was adjusted to 1%.…”