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Cationic antimicrobial peptides are promising sources for novel therapeutic agents against multi-drug-resistant bacteria. HHC-36 (KRWWKWWRR) is a simple but effective antimicrobial peptide with similar or superior activity compared with several conventional antibiotics. In this biophysical study, unique conformational properties of this peptide and some of its analogs as well as its interaction with lipid membranes are investigated in detail. Circular dichroism (CD) and molecular dynamics modeling studies of HHC-36 in different environments reveal a dynamic amphipathic structure composed of competing turn conformations with free energies lower than that of the unfolded state, implying a strong influence of tryptophan interactions in formation of the turns. CD spectra and gel electrophoresis also show strong evidence of self-association of this peptide in aqueous milieu and interaction with both neutrally and negatively charged lipid membrane systems. Isothermal titration calorimetry and acrylamide fluorescence quenching experiments emphasize the preference of HHC-36 for negatively charged vesicles. In addition, dye leakage experiments suggest that this peptide functions through a surface-associated mechanism with weak lytic activity against bacterial model membranes.

McMillan and Mayer (MM) proved two remarkable theorems in their paper on the equilibrium statistical mechanics of liquid solutions. They first showed that the grand canonical partition function for a solution can be reduced to a one with an effectively solute-only form, by integrating out the solvent degrees of freedom. The total effective solute potential in the effective solute grand partition function can be decomposed into components which are potentials of mean force for isolated groups of one, two, three, etc, solute molecules. Secondly, from the first result, now assuming low solute concentration, MM derived an expansion for the osmotic pressure in powers of the solute concentration, in complete analogy with the virial expansion of gas pressure in powers of the density at low density. The molecular expressions found for the osmotic virial coefficients have exactly the same form as the corresponding gas virial coefficients, with potentials of mean force replacing vacuum potentials. In this paper we restrict ourselves to binary liquid solutions with solute species $A$ and solvent species $B$ and do three things: (a) By working with a semi-grand canonical ensemble (grand with respect to solvent only) instead of the grand canonical ensemble used by MM, and avoiding graphical methods, we have greatly simplified the derivation of the first MM result,(b) by using a simple nongraphical method developed by van Kampen for gases, we have greatly simplified the derivation of the second MM result,i.e.,the osmotic pressure virial expansion; as a by-product, we show the precise relation between MM theory and Widom potential distribution theory, and (c) we have extended MM theory by deriving virial expansions for other solution properties such as the enthalpy of mixing. The latter expansion, with changed independent variables corresponding to current experiments, is proving useful., Comment: pdftex, 32 pages, 2 figures. Thermodynamic errata in Section IV.C and Appendix B are corrected. Figure 2 is recalculated at a new temperature to correspond with more recent experimental data

HHC-36 is an antimicrobial peptide, designed through neural network algorithms. It has been tested in vivo and in vitro, and has proved to be strongly effective against strains of multidrug-resistant P. aeruginosa, methicillin-resistant Staphylococcus aureus, and a few other ‘superbugs’ (Cherkasov et al., ACS Chem. Biol., 2009, 4 (1), pp 65-74). The peptide has also been observed through in vivo tests to be greatly pathogen-specific, hence proving to be a great candidate for developing future antibiotics.To understand the mechanism of activity of this peptide against bacterial membranes, we have performed a number of all-atom simulations, together with a series of circular dichroism spectroscopy (CD) and isothermal titration calorimetry (ITC) experiments. The small size (9 amino acids) and great charge density of HHC-36 make it problematic (if not unreliable) to find the structure of HHC-36 through conventional spectroscopy and/or crystallography methods. We have thus performed microsecond-scale molecular dynamics simulations, starting from an unfolded structure of the peptide, to find its folded structure. An amphipathic turn structure has been obtained, which was observed to be very stable over few hundred nanosecond timescales of simulation. This result has been compared to circular dichroism spectroscopy results, and the presence of the turn structure has been verified. To assess the stability of the observed structure, we have also performed temperature-dependent simulations and CD measurements, which have shown the stability of the turn structure at close-to-physiological temperatures.The obtained structure is then used in peptide-membrane simulations with a few different membrane compositions mimicking both bacterial and animal cell membranes. Profiles of the potential of mean force have been obtained, and the relevant binding parameters extracted from these simulations are then compared with the binding free energies obtained from ITC experiments.