Membrane Interactions of Novicidin, a Novel Antimicrobial Peptide:

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Entry by Angelo Mao, AP 225, Fall 2010

Title: Membrane Interactions of Novicidin, a Novel Antimicrobial Peptide: Phosphatidylglycerol Promotes Bilayer Insertion

Authors: Dorosz J, et al

Journal: J. Phys. Chem. B

Volume: Vol 114(34)

Pages: 11053-11060


Novicidin is a novel antimicrobial peptide that is an improvement on certain preexisting antimicrobial peptides (such as melittin) in that it more effectively targets bacteria and causes less hemolysis. Through various experiments, the researchers propose that an explanation for this difference is the relative proportion of hydrophobic to hydrophilic elements in novicidin composition, as well as the locations of these elements, which lowers the free energy of novicidin interaction and embedding in negative (bacterial) membranes in comparison to zwitterionic (human) membranes.

soft matter keywords: phosphilipid, membrane, hydrophobic interactions, hydrophilic interactions, free energy


Figure 1. Schematic of novicidin and interaction with negative parts of the membrane.
Figure 2. Helical wheel projection of novicidin.

Novicidin is a peptide derived from a previous antimicrobial candidate, ovispirin, which unfortunately was highly cytotoxic. As shown in figure 1, novicidin is a generally positively charged peptide. The helical wheel projection of novicidin indicates where the positively charged amino acids, lysine and arginine (in gray), are localized. The hydrophobic residues (green and yellow) are situated on the other side of the wheel, leading to a hydrophobic moment (arrow) in the novicidin molecule. From this, one would expect that novicidin would be able to embed itself in phospholipid membranes with its positive charges facing outside and its hydrophobic elements facing the hydrophobic core of the membrane.

Novicidin incorporation into membranes of varying charge negativities

Figure 3. Isothermal adsorption of novicidin to membrane layers with different proportions of negative groups.
Figure 4. Fluorescence dose−response curves of lipid/PDA vesicles.

The researchers created phospholipid monolayers with varying amounts of a zwitterionic phosopholipid called dimyristoylphosphatidylcholine (DMPG) and negatively charged phospholipid called dimyristoylphosphatidylglycerol(DMPG). The line (iv) in figure 3 indicates the result from the membrane that is entirely composed of DMPG, while the line (i) indicates the result from membrane without any DMPG. Adsorption of novicidin into the membrane increased with DMPG composition, starting from a baseline of near zero adsorption. The researchers performed a similar set of experiments with vesicles of varying amounts of zwitterionic phospholipid/polydiacetylene (PDA) and DMPG. In this other set of experiments, the results of which are shown in figure 4, fluorescent materials leaking from the vesicles were quantified. The results showed positive correlation between DMPG composition and fluorescence intensity. The implication is that novicidin incorporation is not only correlative with the present of negatively charged components in the membrane, but that this incorporation is also correlative to membrane disruption.

Novicidin penetration into membranes of varying charge negativities

Figure 5. Tyrosine fluorescence titration curves.

Novicidin has a tyrosine residue, a fact which the authors utilized in tyrosine fluorescence titration experiments. As shown in figure 5, there was little fluorescence in zwitterionic DMPC vesicles (x in figure 5), but increased fluorescence in vesicles with mixed composition. This indicated very little incorporation of novicidin in DMPC vesicles and increased incorporation in vesicles with DMPG. This experiment seemed primarily to confirm the results of figures 3 and 4, and seems also to be quantitatively inferior, due to the quick saturation of tyrosine.

Novicidin structure changes depending on vesicle composition

Figure 6. Circular dichroism.

Circular dichroism (CD) provides information on molecular structure. The CD for novicidin shows altered structures when in buffer (i) and in the presence of DMPC (ii), from when it is in the present of negatively charged liposomes (iii, iv). The CD implies that structure gained by novicidin in the presence of negatively charged liposomes is alpha-helical from the dips at 208 and 222 nm. The results indicate that incorporation into the bilayer causes novicidin to gain an alpha-helical structure, which is consistent with other antimicrobial proteins.

Novicidin incorporation into membranes leads to comparatively more negative free energy

Figure 7. Free energy.

Based on Monte Carlo simulations, the researchers calculated the free energy from novicidin incorporation into membranes of different amounts of negatively charged elements. In comparison to the more cytotoxic antimicrobial protein melittin, novicidin's free energy interaction with the membrane is more negative with increasing fraction of negatively charged elements in the membrane. This would make novicidin incorporation more favorable. At low fractions of negatively charged elements in the membrane, however, novicidin's free energy of interaction is in fact higher, explaining its decreased cytotoxicity. The researchers also note that novicidin's properties are not explained by its composition alone, but by the distribution of its hydrophobic and hydrophilic elements (figure 2).