Crystallization of S-layers

FIG. 16 Fomation of a Langmuir lipid monolayer at the air/subphase interface and the subsequent crystallization of S-layer protein, (a) Amphiphilic lipid molecules are placed on the air/subphase interface between two barriers. Upon compression between the barriers, increase in surface pressure can be determined by a Wilhelmy plate system, (b) Depending on the final area, a liquid-expanded or liquid-condensed lipid monolayer is formed, (c) S-layer subunits injected in the subphase crystallized into a coherent S-layer lattice beneath the spread lipid monolayer and the adjacent air/subphase interface. 

In the first step, lipid model membranes have been generated (Fig. 15) on the air/liquid interface, on a glass micropipette (see Section VIII.A.1), and on an aperture that separates two cells filled with subphase (see Section VIII.A.2). Further, amphiphilic lipid molecules have been self-assembled in an aqueous medium surrounding unilamellar vesicles (see Section VIII.A.3). Subsequently, the S-layer protein of B. coagulans E38/vl, B. stearother-mophilus PV72/p2, or B. sphaericus CCM 2177 have been injected into the aqueous subphase (Fig. 15). As on solid supports, crystal growth of S-layer lattices on planar or vesicular lipid films is initiated simultaneously at many randomly distributed nucleation... 

The anisotropy in the physicochemical surface properties and differences in the surface topography of S-layer lattices allowed the determination of the orientation of the monolayers with respect to different surfaces and interfaces. Since in S-layers used for crystallization studies the outer surface is more hydrophobic than the inner one, the protein lattices were generally oriented with their outer face against the air-water interface [120,121]. Crystallization studies with the S-layer protein fromB. coagulansE i Nl at different lipid monolayers [122] revealed that the S-layer lattice is attached to lipid monolay-... 

In order to enhance the stability of hposomes and to provide a biocompatible outermost surface shucture for controlled immobihzation (see Section IV), isolated monomeric and oligomeric S-layer protein from B. coagulans E38/vl [118,123,143], B. sphaericus CCM 2177, and the SbsB from B. stearothermophilus PV72/p2 [119] have been crystallized into the respective lattice type on positively charged liposomes composed of DPPC, HD A, and cholesterol. Such S-layer-coated hposomes are spherical biomimetic structures (Fig. 18) that resemble archaeal ceUs (Fig. 14) or virus envelopes. The crystallization of S-... 

Calmano W, Mangold S, Welter E (2001) An XAFS investigation of the artifacts caused by sequential extraction analyses of Pb-contaminated soils. Fresenius J Anal Chem 371 823-830 Carrado KA, Xu L, Gregory D, Song K, Siefert S, Botto RE (2000) Crystallization of a layered silicate clay as monitored by small-angle X-ray scattering and NMR. Chem Mat 12 3052-3059... 

The recrystallization of S-layer proteins at phosphoethanolamine monolayers on aqueous subphases has been also studied on a mesoscopic scale by dual label fluorescence microscopy andFourier transform infrared spectroscopy (FTIR) [110]. It has been shown that the phase state of the lipid exerts a marked influence on the protein crystallization. When the surface monolayer is in the phase separated state between fluid and crystalline phase, the S-layer protein is preferentially adsorbed at the boundary line between the two coexisting phases and crystallization proceeded underneath the crystalline phase. Crystal growth is much slower under the fluid lipid and the entire interface is overgrown only after prolonged protein incubation. In turn, as indicated by characteristic frequency shifts of the methylene stretch vibrations on the lipids, protein crystallization affects the order of the alkane chains and drives the fluid lipid into a state of higher order. However, the protein does not interpenetrate the lipid monolayer as confirmed by x-ray reflectivity studies [105-107]. 

Nature demonstrated the use of regular 2dimensional protein grids in bacterial surface S-layers (5 nm to 15 nm thick). These layers are protein crystals forming the outermost cell envelope of many prokaryotes. A variety of lattice symmetries from pi, p2, p3, p4 to p6 is found in bacterial strains. Sometimes even a single strain is able to switch from one type of S-layer to another induced via a change in the microenvironment. The pores and spacing in between the units varies from 3-30 nm. S-layer proteins are handled dissolved or suspended in a buffer at a concentration of up to 2 mg/ml. Their stability and unique property to coat two-dimensional arrays with perfect uniformity makes them an ideal nano-template. 

Crystal growth of S-layer protein lattices at different surfaces and interfaces was studied by high-resolution electron microscopy and scanning force microscopy (Fig. 14) [20,83]. Generally crystal growth is initiated simultaneously at many randomly distributed nucleation points and proceeds in plane until the crystalline domains meet leading to a closed, coherent mosaic of crystalhne areas... 

Figure 14 High-resolution scanning force microscopical images of S-layers with oblique (pi) (a) and square (p4) lattice symmetry (c) on silicon surfaces. The corresponding computer image reconstructions obtained by cross-correlation averaging are shown in (b) and (d), respectively. Crystal growth is initiated at randomly distributed nucleation points from which crystalline domains grow (e) until the front edges meet and a closed monolayer is formed (f). (Modified from Ref. 89.)...

The predominantly ionic alkali metal sulfides M2S (Li, Na, K, Rb, Cs) adopt the antifluorite structure (p. 118) in which each S atom is surrounded by a cube of 8 M and each M by a tetrahedron of S. The alkaline earth sulfides MS (Mg, Ca, Sr, Ba) adopt the NaCl-type 6 6 structure (p. 242) as do many other monosulfides of rather less basic metals (M = Pb, Mn, La, Ce, Pr, Nd, Sm, Eu, Tb, Ho, Th, U, Pu). However, many metals in the later transition element groups show substantial trends to increasing covalency leading either to lower coordination numbers or to layer-lattice structures. Thus MS (Be, Zn, Cd, Hg) adopt the 4 4 zinc blende structure (p. 1210) and ZnS, CdS and MnS also crystallize in the 4 4 wurtzite modification (p. 1210). In both of these structures both M and S are tetrahedrally coordinated, whereas PtS, which also has 4 4... 

The reaction mixture is poured into a separatory funnel containing about 0 S kg. of crushed ice and is shaken thoroughly. The organic layer is separated, and the aqueous solution is extracted with two SO-ml. portions of methylene chloride. The combined organic solution is washed three times with 75-ml. portions of water A crystal of hydroquinone is added to the methylene chloride solution (Note 1) which is then dried over anhydrous sodium sulfate. After evaporation of the solvent, the residue is distilled to give the crude product, b p. 68-74° (0.9 mm.). After redistillation there is obtained 60-66 g. (81-89%) of mesitaldehyde b.p. 113-115° (11 mm.), 20d 1.5538. 

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