Local stability identification and the role of key acidic amino acid residues in staphylococcal nuclease unfolding.

Staphylococcal nuclease is a single domain protein with 149 amino acids. It has no disulfide bonds, which makes it a simple model for the study of protein folding. In this study, 20 mutants of this protein were generated each with a single base substitution of glycine for negatively charged glutamic acid or aspartic acid. Using differential scanning microcalorimetry in thermal denaturation experiments, we identified two mutants, E75G and E129G, having approximately 43% and 44%, respectively, lower DeltaH(cal) values than the wild-type protein. Furthermore, two mutants, E75Q and E129Q, were created and the results imply that substitution of the Gly residue has little influence on destabilization of the secondary structure that leads to the large perturbation of the tertiary protein structure stability. Two local stable areas formed by the charge-charge interactions around E75 and E129 with particular positively charged amino acids are thus identified as being significant in maintenance of the three-dimensional structure of the protein.

Induction of transient ion channel-like pores in a cancer cell by antibiotic peptide.

The anticancer activity of anti-bacterial cecropins makes them potentially useful as peptide anti-cancer drugs. We used the cell-attached patch to study the effect of cecropin B (CB; having one hydrophobic and one amphipathic alpha-helix) and its derivative, cecropin B3 (CB3; having two hydrophobic alpha-helices) on the membrane of Ags cancer cells. Application of 10-60 microM CB onto the membrane of the cancer cell produces short outward currents. Comparative study with CB3, which induces no outward currents, shows that the amphipathic group of CB is necessary for the pore formation. The results provide a rationale to study the cell-killing activity of antimicrobial peptides at the single cancer cell level.

Distinguishing between different pathways of bilayer disruption by the related antimicrobial peptides cecropin B, B1 and B3.

Different pathways of bilayer disruption by the structurally related antimicrobial peptides cecropin B, B1 and B3, revealed by surface plasma resonance analysis of immobilized liposomes, differential scanning calorimetry of peptide-large unilamellar vesicle interactions, and light microscopic analysis of peptide-treated giant unilamellar vesicles, have been identified in this study. Natural cecropin B (CB) has one amphipathic and one hydrophobic alpha-helix, whereas cecropins B1 (CB1) and B3 (CB3), which are custom-designed, chimaeric analogues of CB, possess either two amphipathic or two hydrophobic alpha-helices, respectively. Surface plasma resonance analysis of unilamellar vesicles immobilized through a biotin-avidin interaction showed that both CB and CB1 bind to the lipid bilayers at high concentration (>10 microm); in contrast, CB3 induces disintegration of the vesicles at all concentrations tested. Differential scanning calorimetry showed the concentration-dependent effect of bilayer disruption, based on the different thermotrophic phase behaviours and the shapes of the thermal phase-transition curves obtained. The kinetics of the lysis of giant unilamellar vesicles observed by microscopy demonstrated that both CB and CB1 effect a continuous process involving loss of integrity followed by coalescence and resolution into smaller vesicles, whereas CB3 induces rapid formation of irregular-shaped, nonlamellar structures which rapidly disintegrate into twisted, microtubule-containing debris before being completely destroyed. On the basis of these observations, models by which CB, CB1 and CB3 induce lysis of lipid bilayers are discussed.