Protein crystallization lipidation

In this chapter we describe the basic principles involved in the controlled production and modification of two-dimensional protein crystals. These are synthesized in nature as the outermost cell surface layer (S-layer) of prokaryotic organisms and have been successfully applied as basic building blocks in a biomolecular construction kit. Most importantly, the constituent subunits of the S-layer lattices have the capability to recrystallize into iso-porous closed monolayers in suspension, at liquid-surface interfaces, on lipid films, on liposomes, and on solid supports (e.g., silicon wafers, metals, and polymers). The self-assembled monomolecular lattices have been utilized for the immobilization of functional biomolecules in an ordered fashion and for their controlled confinement in defined areas of nanometer dimension. Thus, S-layers fulfill key requirements for the development of new supramolecular materials and enable the design of a broad spectrum of nanoscale devices, as required in molecular nanotechnology, nanobiotechnology, and biomimetics [1-3]. 

It has been shown by FM that the phase state of the lipid exerted a marked influence on S-layer protein crystallization [138]. When the l,2-dimyristoyl-OT-glycero-3-phospho-ethanolamine (DMPE) surface monolayer was in the phase-separated state between hquid-expanded and ordered, liquid-condensed phase, the S-layer protein of B. coagulans E38/vl was preferentially adsorbed at the boundary line between the two coexisting phases. The adsorption was dominated by hydrophobic and van der Waals interactions. The two-dimensional crystallization proceeded predominately underneath the liquid-condensed phase. Crystal growth was much slower under the liquid-expanded monolayer, and the entire interface was overgrown only after prolonged protein incubation. 

Membrane integral proteins have transmembrane domains that insert directly into lipid bilayers. Transmembrane domains (TMDs) consist predominantly of nonpolar amino acid residues and may traverse the bilayer once or several times. High-resolution structural information is available for only a few integral membrane proteins, primarily because it is difficult to obtain membrane protein crystals that are adequate for X-ray diffraction measurements. 

Ai, X., Caeerey, M., Membrane protein crystallization in lipidic mesophases Detergent effects. Biophys. J. 2000, 79(1), 394-405. 

Lipid cubic (51) and sponge (52) phases, as well as bicelles (53), are alternatives to detergents that have been applied successfully to membrane protein crystallization. In these instances, the protein is embedded in a lipid bilayer environment, which is considered more natural compared with the detergents that form micellar phases. In the recent high-resolution crystal structure of the human 32 adrenergic G-protein-coupled receptor, lipid cubic phase was used with necessary cholesterol and 1,4-butandiol additives (54). The cholesterol and lipid molecules were important in facilitating protein-protein contacts in the crystal. 

Protein crystallization, NMR, FTIR and AFM studies usually required large quantities of homogeneous proteins. Chemical approaches as shown above allow for production of reasonable amount of lipid modified proteins with well-defined structures as well as incorporation of reporter groups into proteins. These strategies have profoundly facilitated the structural, biophysical and cellular studies of the function of lipidated proteins. Some examples are discussed in this section. 

It is very difficult to directly observe water molecules in vivo at the surfaces of proteins or lipid bilayers or in the grooves of DNA. X-ray studies of protein crystals cannot provide full information as only a fi action of water molecules remain on the surface and even then in restricted positions. Also, the hydration layer is mobile in solution. One would like to know about the structural and dynamical characteristics of these water molecules in the active state, ideally within biological cells. Such detailed information is still not available in most cases. Much of our current understanding of water in biological systems has come from study of proteins and DNA in aqueous solution. 

The use of so-called S-layers is a combination of self-organization and spatial patterning [88]. S-layers consist of 2-D protein crystals that are formed naturally as the outermost cell surface layer (S-layer) of prokaryotic organisms. The subunits can recrystallize into nanoporous monolayers in suspension, at liquid-surface interfaces, on lipid films, or on solid substrates. The S-layers of Bacillus sphaericus CCM 2177 have been used to generate ordered arrays of 4—5 nm gold particles, with a 13.1 nm repeat distance, from AuCr ions [89]. The spontaneous self-assembly of 5 nm AuN Ps was shown to occur at the S-layer of Deinococcus radiodurans, to produce micrometersized ordered domains [90]. Arrays of 1.9 nm platinum particles were achieved from Pt salts in the S-layer of Sporosarcina ureae [90] these were of square symmetry and had a lattice constant of 13.2 nm. 

Essentially the same consideration appUes to an inverted bicontinuous cubic phase (Qn) and an inverted hexagonal (Hn) phase, which are usually formed with lipids with long hydrophobic chains. Qn and Hn phases have recently received growing attention in the pharmaceutical or biological fields, for instance, as new carriers for drug-deUvery systems, and matrices for membrane protein crystallization [37-42]. The conventional (e.g., monoolein) Qn phase, however, often transforms into a soUd phase at low temperatures around 4°C [43-47], where temperature-sensitive proteins or actives are most preferably handled and preserved. It has recently been confirmed that isoprenoid-chained lipids can in fact give a range of Qn phases that are stable at low temperatures [13]. 

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