Analogues of cholesterol (compounds 1 and 2) and coprostanol (compound 3) containing the BODIPY fluorophore in the aliphatic tail of the free sterol have been synthesized starting with bisnorcholenic acid, cholenic acid 3beta-acetate, and lithocholic acid, respectively. An ester linkage joining the fluorophore to the sterol nucleus interfered with the ability of the fluorescent sterol to pack with phospholipids in monolayers. However, an analogue in which the linker was devoid of polar atoms exhibited a substantially similar physical behavior to cholesterol in model membranes with respect to localization in raft domains.
Daptomycin and A54145 are homologous lipopeptide antibiotics that permeabilize the cell membranes of Gram-positive bacteria. Membrane permeabilization depends on the presence of both phosphatidylglycerol (PG) and calcium, and it involves the formation of oligomeric transmembrane pores that consist of approximately 6-8 subunits. We here show that each lipopeptide molecule binds two calcium ions in separable, successive steps. The first calcium ion causes the lipopeptide molecule to bind to the target membrane, and likely to form a loosely associated oligomer. Higher calcium concentrations induce binding of a second ion, which produces the more tightly associated and more deeply membrane-inserted final, functional form of the oligomer. Both calcium-dependent steps are accompanied by fluorescence signals that indicate transition of specific amino acid residues into less polar environments, suggestive of insertion into the target membrane. Our findings agree with the earlier observation that two of the four acidic amino acid residues in the daptomycin molecule are essential for antibacterial activity.
Daptomycin is a clinically important lipopeptide antibiotic that kills Gram-positive bacteria through membrane depolarization. Its activity requires calcium and the presence of phosphatidylglycerol in the target membrane. Calcium and phosphatidylglycerol also promote the formation of daptomycin oligomers, which have been assumed but not proven to be required for the bactericidal effect. Daptomycin shares substantial structural similarity with another lipopeptide antibiotic, A54145; the two have identical amino acid residues in 5 out of 13 positions and similar ones in 4 more positions. We here examined whether these conserved residues are sufficient for oligomer formation. To this end, we used fluorescence energy transfer and excimer fluorescence to detect hybrid oligomers of daptomycin and CB-182,462, a semisynthetic derivative of A54145. Mixtures of the two compounds indeed produced hybrid oligomers, but at the same time displayed a significantly less than additive antibacterial activity against Bacillus subtilis. The existence of functionally impaired oligomers indicates that oligomer formation is indeed important for antibacterial function. However, it also shows that oligomerization is not sufficient; once formed, the oligomers must take another step in order to acquire antibacterial activity. Thus, the amino acid residues shared between daptomycin and CB-182,462 suffice for formation of the oligomer, but not for its subsequent activation.
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