Measurement of the affinity of melittin for zwitterionic and anionic membranes using immobilized lipid biosensors.
The binding of melittin to zwitterionic dimyristyphosphatidylcholine (DMPC) and anionic dimyristylphosphatidylglycerol (DMPG) was analysed using two different immobilized model membrane systems. The first system used surface plasmon resonance (SPR), which monitors the real-time binding of peptides to an immobilized hybrid bilayer. SPR experiments reflected a stronger binding of melittin for DMPG than for DMPC, while kinetic analysis suggested the existence of at least two distinct binding steps. The second lipid biosensor system involved an immobilized phospholipid monolayer covalently attached to a microporous silica surface. The binding of melittin to the immobilized monolayer was then monitored using dynamic elution chromatography with varied methanol concentrations to analyse the binding of melittin to DMPC and DMPG. The nonlinear binding behaviour observed for melittin with the phosphatidylcholine (PC) and phosphatidylglycerol (PG) monolayers compared with the linear retention plots and Gaussian peak shapes observed for the control molecule demonstrated that melittin undergoes significant conformational and orientational changes upon binding to the immobilized PC and PG ligands. The dependence of log k' on per cent methanol also demonstrated a bimodal interaction whereby hydrophobic forces predominated at higher temperatures and methanol concentrations, while other forces, presumably electrostatic in nature, also made a contribution to the affinity of the peptides for the lipid monolayer, particularly at lower temperatures. The complementary use of these two lipid biosensors thus allows the role of hydrophobic and electrostatic forces in peptide-membrane interactions to be studied.
Lee
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TH
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
Mozsolits
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H
Aguilar
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MI
D000838:Anions; D015374:Biosensing Techniques; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002942:Circular Dichroism; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry / Q000378:metabolism; D008051:Lipid Bilayers; D050356:Lipid Metabolism; D008555:Melitten / Q000737:chemistry* / Q000378:metabolism*; D010715:Phosphatidylglycerols / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D020349:Surface Plasmon Resonance
D000838:Anions; D008051:Lipid Bilayers; D010715:Phosphatidylglycerols; D008555:Melitten; C002773:dimyristoylphosphatidylglycerol; D004134:Dimyristoylphosphatidylcholine