title: Binding of peptides corresponding to the carboxy-terminal region of human-β-defensins-1-3 with model membranes investigated by isothermal titration calorimetry.
abstract: Human-β-defensins HBD-1-3 are important components of the innate immune system. Synthetic peptides Phd-1-3 with a single disulphide bond, spanning the cationic C-terminal region of HBD-1-3, have antimicrobial activity. The interaction of Phd-1-3 with model membranes was investigated using isothermal titration calorimetry (ITC) and steady-state fluorescence polarization to understand the biophysical basis for the mechanism of antimicrobial action. Calorimetric titration of POPE:POPG (7:3) vesicles with peptides at 25°C and 37°C showed complex profiles with two distinct regions of heat changes. The data indicate binding of Phd-1-3 at 37°C to both negative and zwitterionic lipid vesicles is exothermic with low enthalpy values (ΔH~-1.3 to -2.8kcal/mol) as compared to amphipathic helical antibacterial peptides. The adsorption of peptides to negatively charged lipid membranes is modulated by electrostatic interactions that are described by surface partition equilibrium model using Gouy-Chapman theory. However, this model could not explain the isotherms of peptide binding to zwitterionic lipid vesicles. Fluorescence polarization of TMA-DPH (1-[4-(trimethylammonio) phenyl]-6-phenyl-1,3,5-hexatriene) and DPH (1,6-diphenyl-1,3,5-hexatriene) located in the head group and acyl chain region respectively, indicates that the peptides interact with interfacial region of negatively charged membranes. Based on the results obtained, we conclude that adsorption of cationic peptides Phd-1-3 on lipid surface do not result in conformational change or pore formation. It is proposed that interaction of Phd-1-3 with the negatively charged lipid head group causes membrane destabilization, which in turn affects the efficient functioning of cytoplasmic membrane proteins in bacteria, resulting in cell death.
authors.affiliation: CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India. krishnak@ccmb.res.in
authors.forename: V
authors.initials: V
authors.lastname: Krishnakumari
authors.forename: R
authors.initials: R
authors.lastname: Nagaraj
mesh_terms: D000327:Adsorption; D001419:Bacteria / Q000737:chemistry; D002153:Calorimetry, Indirect / Q000379:methods; D004095:Dihydropyridines / Q000737:chemistry; D004161:Diphenylhexatriene / Q000031:analogs & derivatives / Q000737:chemistry; D005454:Fluorescence Polarization / Q000379:methods; D006801:Humans; D008567:Membranes, Artificial; D010713:Phosphatidylcholines / Q000737:chemistry*; D010714:Phosphatidylethanolamines / Q000737:chemistry*; D023083:beta-Defensins / Q000737:chemistry*
chemical_list: C466879:2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylic acid monomethyl ester; C406407:DEFB1 protein, human; C106641:DEFB4A protein, human; D004095:Dihydropyridines; D008567:Membranes, Artificial; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D023083:beta-Defensins; C057561:1-palmitoyl-2-oleoylphosphatidylethanolamine; D004161:Diphenylhexatriene; C033628:1-(4-(trimethylamino)phenyl)-6-phenylhexa-1,3,5-triene; C028694:1-palmitoyl-2-oleoylphosphatidylcholine