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The research in my group focuses on protein-lipid interaction in biological membranes. Main topics are:

Our experiments involve a range of methods - fluorescence, protein chemistry, and molecular biology. The results of this work are of both theoretical and medical interest. Recent examples include:

Interaction of the lipopeptide antibiotic daptomycin with bacterial membranes
Detection of daptomycin oligomerization by FRET

The lipopeptide antibiotic daptomycin is active against Gram-positive bacteria and is important in the treatment of infections. It binds to the bacterial cell membranes and causes depolarization. While it had long been surmised that this process might involve the formation of membrane-associated oligomers, this had not been proven.

The daptomycin molecule contains a kynurenine residue, whose fluorescence can be observed both on liposomes (plot on the right) and bacterial cells. It also contains a unique amino group, to which a second fluorophore, NBD, can be attached. A mixture of unlabeled and NBD-labeled daptomycin can then be used to detect oligomer formation by fluorescence energy transfer (FRET) from kynurenine to NBD, which is evident from a sharp drop in kynurenine fluorescence. If the two species are applied sequentially rather than simultaneously, some FRET is observed also, which occurs most likely between oligomers, rather than within.

Using this and some other fluorescence techniques, we have since shown that daptomycin the oligomer contains approximately 6–7 subunits, that it is necessary for antibacterial activity, and that its formation involves a stoichiometric interaction with phosphatidylglycerol (PG). Further investigations are ongoing.

Muraih JK, Palmer M. Estimation of the subunit stoichiometry of the membrane-associated daptomycin oligomer by FRET. Biochim. Biophys. Acta (2012) 1818:1642-1647

Muraih, J.K., Harris, J., Taylor, S.D, Palmer, M.: Characterization of daptomycin oligomerization with perylene excimer fluorescence: Stoichiometric binding of phosphatidylglycerol triggers oligomer formation (2012). Biochim. Biophys. Acta 1818:673-678

Muraih, J.K., Pearson, A., Silverman, J., Palmer, M.: Oligomerization of daptomycin on membranes (2011). Biochim. Biophys. Acta 1808:1154-60

Zhang, T., Muraih, J.K., Mintzer, E., Tishbi, N., Desert, C., Silverman, J., Tayler, S., Palmer, M.: Mutual inhibition through hybrid oligomer formation of daptomycin and the semisynthetic lipopeptide antibiotic CB-182,462 (2012). Biochim. Biophys. Acta (in press)

This page contains the literature that I have collected on daptomycin and related lipopeptides.

A small, inactive fragment of the pore-forming toxin pyolysin amplifies the activity of the intact wild-type toxin molecule
Acceleration of hemolysis by the pyolysin domain 4 fragment

Pyolysin is a protein toxin that punches holes into membranes, similar to those shown below for CAMP factor, only larger. The C-terminal domain 4 had been assumed to be involved in membrane binding of the toxin, but otherwise to do little else.

Hybrid oligomers of wt pyolysin and the d4 fragment

However, as you can see in the image on the right, the domain 4 (d4) fragment does do more. On its own, it does not do anything (solid line); however, when added to a fixed amount of wild-type toxin (wt), it increases the activity of the latter, as evident from the accelerated destruction of red blood cells (whose concentration is here measured by way of turbidity).

An interaction of the fragment and wild type pyolysin can also be observed in the electron microscope (on the right). When applied to crystalline cholesterol (a membrane surrogate) on its own, the wild type toxin forms rings, whereas the fragment forms straight rods; a mixture forms walking cane-like structures.

Pokrajac, L., Harris, J.R., Sarraf, N., Palmer, M. Oligomerization and Hemolytic Properties of the C-Terminal Domain of Pyolysin, a Cholesterol Dependent Cytolysin. Biochemistry and Cellular Biology (2012), in press

Dual targeted labeling of proteins using cysteine and
selenomethionine residues
Reaction of selenomethionine with iodoacetamide

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.

Streptococcus agalactiae CAMP factor binds to GPI-anchored proteins
Calcein release by liposomes with PLAP

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.

Wu, X., Shen, Z., Zeng, X., Lang, S., Palmer, M, Guo, Z. Synthesis and biological evaluation of sperm CD52 GPI anchor and related derivatives as binding receptors of pore-forming CAMP factor (2008). Carbohydr Res. 343:1718-29.

Streptococcus agalactiae CAMP factor (protein B) is a pore-forming toxin
Electron microscopy of CAMP factor holes

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.

The 'cholesterol-binding cytolysins' are ambidextrous
Dose-effect curves of SLO on liposomes with cholesterol and ent-cholesterol

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

Chloramphenicol acts in the cytosol - so why would E.coli care to remove it from the periplasm?
Schematic of drug extrusion in gram-negative bacteria

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