Lipid attachment

Although ribosomal proteins are readily observed as in Figures 13.7 and 13.8 altered matrix conditions can alter the relative ionization of bacterial whole-cell compounds. A systematic analysis involving laser power/fluence and sample preparation conditions reveals that if the concentrated trifluo-roacetic acid is added and the laser power increased above optimal conditions, ionization of bacterial surface compounds can be enhanced. Figure 13.9 is the resulting 9.4 T MALDI-FTMS, seen are both the Braun s lipoprotein56,57 and the Murein lipoprotein. Both of these compounds are complex combinations of hydrocarbon lipids attached to a protein base. This is the first MALDI-FTMS observation of surface proteins desorbed directly from whole cells by influencing ionization conditions. 

Traditionally, prokaryotic expression, especially employment of E. coli-based vectors, has been the system of choice. However, bacteria are unable to provide many vital components required for post-translational modifications including various forms of glycosyla-tion or lipid attachment and protein processing, all of which can also be important for proper protein folding. For this reason, it is not surprising that much time and effort has been dedicated to the development of alternative systems, summarized in Tab. 1.2. 

Of the extremely diverse examples of protein modifications observed in eukaryotic cells, the modifications by lipid (and glycolipid) molecules are of special interest because lipid-attached proteins can be anchored at the membrane, although all of these proteins are not always anchored. So far, three groups of membrane anchoring proteins have been noted (Fig. 5). 

To enable lipid attachment, repeat steps 3-6 using (Fmoc)-K(Mtt)-OH to enable lipid attachment between the two epitopes. 

Repeat steps 3-6 using (Boc)-Gly-OH to temporarily block the N-terminus of the peptide. The Boc-protective group is resistant to removal by the conditions used for lipid attachment until cleavage of the assembled product from the resin and concomitant removal of the side-chain-protecting groups. 

Repeat steps 2-6 in order to couple two serines to the exposed e-amino group of the intervening lysine residue and remove the Fmoc group from the second serine residue. The peptide is now ready for lipid attachment (see Subheading 3.2.),... 

PROSITE PS00013 201 211 Membrane lipprot.lipid attachment site... 

Body cells have a membrane that is quite complicated but it ean be represented by a double layer of lipids or lipids attached to proteins. Thus the behavior of lipids and lipid-like molecules becomes very important in un-... 

Lipids that are found associated with proteins go by the term lipoproteins. Lipids attached to sugars or carbohydrates are called glycolipids. There are also lipids attached to alcohols and some to phosphoric acids. The attachment with other compounds greatly alters the behav-... 

Jing S, Trowbridge IS. 1987. Identification of the intermolecular disulfide bonds of the human transferrin receptor and its lipid-attachment site. EMBO J. 6 327-31... 

Long-chain lipids attached to certain amino acids anchor some proteins to one or the other membrane leaflet (see Figure 5-15). 

In the Eastern-Western procedure, lipids are first applied to a solid support such as nitrocellulose. Next, proteins subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis are transferred by standard Western blotting techniques to the solid support in such a manner that the protein of interest is transferred to the lipid patch. During electrotransfer of protein to the solid support, protein, lipid, and sodium dodecyl sulfate mix and as transfer continues the sodium dodecyl sulfate is removed leaving behind the protein to refold in the presence of lipid. Attachment of the refolded protein to a solid support allows one to probe protein structure using conformation-sensitive antibodies or protein function... 

The function of these additional chemical groups is not well understood. In some cases, they serve as molecular switches, activating the protein only when it is needed. Glycosolation is the most prevalent modification in eukaryotic cells, and it appears to increase the protein s solubility and decrease its susceptibility to proteolytic attack. Lipid attachment to proteins is usually seen when the protein s function requires its presence near a membrane surface within the cell. The polar portion of the lipid is attached covalently to the protein while the hydrophobic tail of the lipid is imbedded in the membrane. The lipid group thus serves as an anchor for the soluble protein. Finally, many of the enzymes... 

Scheme 15 Site-specific lipid attachment through sortase-mediated transpeptidation. (a) Mechanism of sortase-mediated ligation, (b) Semisynthesis of lipid modified GFP protein by sortase-mediated ligation, (c) Semisynthesis of lipidated K-Ras4B protein, (d) Semisynthesis of GPI modified GFP protein...

DOPA-derivatized polymerizable lipids have recently been reported. The diacetylenic monomer VII contains a hydro-phobic polymerizable lipid attached to a DOPA-containing polar head group. Self-assembly of VII into vesicles and light-induced polymerization yielded stable adhesive vesicles, fibrils, and sheets, and the interfacial adhesive behavior of these stmaures was evaluated. 

Although PrP presses a highly conserved hydro-phobic domain, it is not an integral membrane protein, but it is anchored to cell surface plasma membranes by a glycosylphospatidylinositol (GPI) (see Membrane lipids) attached to its C-terminal amino acid. This GPI is unusual in that it is sialylated. PrP (the normal isoform of the protein) is completely degraded by proteases... 

Finally, note that the overspill effect is not specific to rod-coil aggregates. It is expected to play a role in the phase separation of membranes incorporating both monomeric lipids and polymerized lipids or lipids attached to a hydrophilic chain. 

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