For structure-function studies of proteins, it is useful to be able to introduce two different labels to defined residues in a single protein molecule. While introducing the first label using a cysteine residue is straightforward, attachment of the second label is more challenging. We have developed a strategy to do that using selenomethionine as the target site for the second label. The label may have a iodoacetamide reactive group or, alternatively, a benzyl bromide group.

We are currently exploring the use of this methodology for studying the activity of pore-forming toxins by fluorescence energy transfer.

Lang, S., Spratt, D.E., Guillemette, J.G., Palmer, M (2005): Dual-targeted labeling of proteins using cysteine and selenomethionine residues. Anal. Biochem. (2005): 342, 271-9.

Lang S, Spratt DE, Guillemette JG, Palmer M. (2006): Selective labeling of selenomethionine residues in proteins with a fluorescent derivative of benzyl bromide. Anal Biochem. 359:253-8.

CAMP factor is a bacterial toxin that forms pores in membranes (see below). Its activity on cell membranes is much higher than on model membranes such as liposomes. This is because it binds to specific molecules on cell membranes - the carbohydrate moieties of GPI anchors.

The image shows that liposomes can be made more sensitive, too, if a GPI-anchored protein is incorporated into their membranes. Liposomes carrying the protein - alkaline phosphatase, PLAP - release a fluorescent marker when treated with CAMP factor, whereas those without do not. If PLAP is removed with a phospholipase that cleaves GPI anchors (PI-PLC), the susceptibility drops sharply.

We want to investigate the interaction of GPI anchors and CAMP factor in more detail - this project would be suitable for a future graduate student.

Lang S, Xue J, Guo Z, Palmer M. (2006): Streptococcus agalactiae CAMP factor binds to GPI-anchored proteins. Med Microbiol Immunol (Berl), epub.

Here, you can see two holes the toxin punched into the membrane of a red blood cell. The holes are very likely lined by a thin seam of oligomeric CAMP factor protein.

One of our present aims is to elucidate the conformation of the CAMP factor molecules within this pore structure. We will try to work this out using a variety of biochemical and biophysical techniques, including mutagenesis, fluorescence and NMR.

Lang, S., Palmer, M. (2003): Characterization of Streptococcus agalactiae CAMP factor as a pore-forming toxin. J. Biol. Chem., 278:38167-73.

Bacterial pore-forming toxins must be specific for animal as opposed to bacterial cell membranes. With streptolysin O, this is accomplished by a requirement for membrane cholesterol, and SLO and and its homologous toxins are frequently called 'the cholesterol-binding cytolysins'.

Here, however, you can see that enantiomeric cholesterol (ent-cholesterol) is only slightly less effective than cholesterol in sensitizing membranes to SLO, suggesting that the recognition of the sterol by SLO is not structurally very specific. This just illustrates how elusive protein-lipid interaction frequently is - there is a lot to be understood and discovered in this area.

Zitzer, A., Westover, E.J., Covey, D.F., Palmer, M. (2003): Differential interaction of the two cholesterol-dependent, membrane-damaging toxins, streptolysin O and Vibrio cholerae cytolysin, with enantiomeric cholesterol. FEBS Lett.,553:229-31

One mechanism of drug resistance in bacteria consists in just expelling the drug molecules from the cell; this is accomplished by active transporters in the cell membrane. Gram-negative cells have two membranes, and there are transporters for extrusion across either of them. This even holds for cytoplasmically acting drugs like tetracycline and chloramphenicol, which are thus extruded in a sequential fashion, rather than in a single step across both membranes.

Why is this? A theoretical model shows that the sequential export is critically more effective than a direct export across both membranes could be.

Palmer, M. (2003): Efflux of cytoplasmically acting antibiotics from gram-negative bacteria: Periplasmic substrate capture by multi-component efflux pumps inferred from their cooperative action with single-component transporters. J. Bacteriol. 185, 5287-9