Gramicidin C

We have measured the lateral diffusion coefficient (D), of active dansyl-labeled gramicidin C (DGC), using the technique of fluorescence photobleaching recovery, under conditions in which the cylindrical dimer channel of DGC predominates. In pure, hydrated, dimyristoylphosphatidylcholine (DMPC) multibilayers (MBL), D decreases from 6 X 10(-8) cm2/s at 40 degrees C to 3 X 10(-8) cm2/s at 25 degrees C, and drops 100-fold at 23 degrees C, the phase transition temperature (Tm) of DMPC. Above Tm, addition of cholesterol decreases D; a threefold stepwise drop occurs between 10 and 20 mol %. Below Tm, increasing cholesterol increases D; a 10-fold increase occurs between 10 and 20 mol % at 21 degrees C, between 20 and 25 mol % at 15 degrees C, and between 25 and 30 mol % at 5 degrees C. In egg phosphatidylcholine (EPC) MBL, D decreases linearly from 5 X 10(-8) cm2/s at 35 degrees C to 2 X 10(-8) cm2/s at 5 degrees C; addition of equimolar cholesterol reduces D by a factor of 2. Thus this transmembrane polypeptide at low membrane concentrations diffuses quite like a lipid molecule. Its diffusivity in lipid mixtures appears to reflect predicted changes of lateral composition. Increasing gramicidin C (GC) in DMPC/GC MBL broadened the phase transition, and the diffusion coefficient of the lipid probe N-4-nitrobenzo-2-diazole phosphatidylethanolamine (NBD-PE) at 30 degrees C decreases from 8 X 10(-8) cm2/s below 5 mol % GC to 2 X 10(-8) cm2/s at 14 mol % GC; D for DGC similarly decreases from 4 X 10(-8) cm2/s at 2 mol % GC to 1.4 X 10(-8) cm2/s at 14 mol % GC. Hence, above Tm, high concentrations of this polypeptide restrict the lateral mobility of membrane components.

Antimicrobial peptides (AMPs) are promising antibacterial agents often hindered by their undesired hemolytic activity. Inspired by gramicidin S (GS), a well-known cyclodecapeptide, we synthesized a panel of antibacterial cyclopeptidomimetics using β,γ-diamino acids (β,γ-DiAAs). We observed that peptidomimetic CP-2 displays a bactericidal activity similar to that of GS while possessing lower side-effects. Moreover, extensive studies revealed that CP-2 likely kills bacteria through membrane disruption. Altogether, CP-2 is a promising membrane-active antibiotic with therapeutic potential.

Gramicidin channels are mini-proteins composed of two tryptophan-rich subunits. The conducting channels are formed by the transbilayer dimerization of nonconducting subunits, which are tied to the bilayer/solution interface through hydrogen bonds between the indole NH groups and the phospholipid backbone and water. The channel structure is known at atomic resolution and the channel's permeability characteristics are particularly well defined: gramicidin channels are selective for monovalent cations, with no measurable permeability to anions or polyvalent cations; ions and water move through a pore whose wall is formed by the peptide backbone; and the single-channel conductance and cation selectivity vary when the amino acid sequence is varied, even though the permeating ions make no contact with the amino acid side chains. Given the amount of experimental information that is available--for both the wild-type channels and for channels formed by amino acid-substituted gramicidin analogues--gramicidin channels provide important insights into the microphysics of ion permeation through bilayer-spanning channels. For the same reason, gramicidin channels constitute the system of choice for evaluating computational strategies for obtaining mechanistic insights into ion permeation through the complex channels formed by integral membrane proteins.