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 <1min, was followed by a slower step with a half-life of >3min. Conversion between the states was estimated to have an enthalpy of activation of approximately 10kJmol−1 and an entropy of activation of −0.2kJKmol−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.
Highlights
Defensins are arginine-rich cationic peptides with molecular weights of 3–5 kDa which adopt predominantly β-sheet structures stabilised by 3–4 disulfide bonds [1,2,3]
The equilibrium constant for human neutrophil peptide (HNP)-2 dimerisation was determined by fluorescence spectroscopy in order that the aqueous-state oligomeric form of the peptide was known for membrane binding experiments
A series of fluorescence measurements was made in water on solutions of human neutrophil peptide-2 (HNP-2) in order to study the self-association of the peptide
Summary
Defensins are arginine-rich cationic peptides with molecular weights of 3–5 kDa which adopt predominantly β-sheet structures stabilised by 3–4 disulfide bonds [1,2,3]. HNP-1–3 have primary structures that only differ by a single residue at the N-terminus [5] These defensins adopt a three-stranded antiparallel β-sheet structure [6] stabilised by six cysteine residues, paired Cys1–Cys, Cys2–Cys and Cys3–Cys (where the numbers reflect the relative position in the sequence, not residue numbers) [7,8]. 20% binding of HNP-2 was observed with 10 mol% POPG at a total lipid concentration of 10 mM, increasing to approximately 60% with 20 mol% POPG. In this paper we determine the kinetics of HNP-2 association with membranes using fluorescence and linear dichroism spectroscopies, combined with studies on the aggregation behaviour of the liposomes in the presence of the peptide. From the LD data in particular, we are able to analyse the alignment of the membrane-bound peptide and assess the binding models proposed in the literature
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