title: The association of defensin HNP-2 with negatively charged membranes: A combined fluorescence and linear dichroism study.
abstract: The association of defensin HNP-2 with negatively charged membranes has been studied using a new approach that combines fluorescence and linear dichroism (LD) spectroscopies with simulated LD spectra in order to characterise the binding kinetics and bound configurations of the peptide. Binding to membranes composed of mixtures of diacylglycerophosphocholines (PC) with either diacylglycerophosphoglycerol (PG) or diacylglycerophosphoserine (PS) was conducted at lipid:peptide ratios that yielded binding, but not membrane fusion. HNP-2 association with membranes under these conditions was a 2 stage-process, with both stages exhibiting first order kinetics. The fast initial step, with a half-life of < 1 min, was followed by a slower step with a half-life of > 3 min. Conversion between the states was estimated to have an enthalpy of activation of approximately 10 kJ mol(-1) and an entropy of activation of -0.2 kJ K mol(-1). LD spectra corresponding to each of the membrane bound states were generated by non-linear regression using a standard kinetic model. These spectra are interpreted in comparison with spectra calculated using the program Dichrocalc and reveal that the peptide associates with membranes in a small number of stable configurations. All of these configurations have a significant proportion of β-sheet structure residing in the plane of the membrane. Two configurations support structures previously proposed for defensins in membranes.
authors.affiliation: Chemistry Department, Durham University, Durham DH1 3LE, UK.
authors.forename: Catherine J
authors.initials: CJ
authors.lastname: Pridmore
authors.affiliation: Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Alison
authors.initials: A
authors.lastname: Rodger
authors.affiliation: Chemistry Department, Durham University, Durham DH1 3LE, UK. Electronic address: j.m.sanderson@durham.ac.uk.
authors.forename: John M
authors.initials: JM
authors.lastname: Sanderson
mesh_terms: D002942:Circular Dichroism; D023082:Defensins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008566:Membranes / Q000737:chemistry*; D017433:Protein Structure, Secondary; D013816:Thermodynamics
chemical_list: D023082:Defensins; D008051:Lipid Bilayers; C556637:neutrophil defensin hNP2, human
keywords: Antibiotic peptide; Binding; Fluorescence; Kinetics; Linear dichroism; Lipid membrane