title: Control and role of pH in peptide-lipid interactions in oriented membrane samples.
abstract: To understand the molecular mechanisms of amphiphilic membrane-active peptides, one needs to study their interactions with lipid bilayers under ambient conditions. However, it is difficult to control the pH of the sample in biophysical experiments that make use of mechanically aligned multilamellar membrane stacks on solid supports. HPLC-purified peptides tend to be acidic and can change the pH in the sample significantly. Here, we have systematically studied the influence of pH on the lipid interactions of the antimicrobial peptide PGLa embedded in oriented DMPC/DMPG bilayers. Using solid-state NMR (31P, 2H, 19F), both the lipid and peptide components were characterized independently, though in the same oriented samples under typical conditions of maximum hydration. The observed changes in lipid polymorphism were supported by DSC on multilamellar liposome suspensions. On this basis, we can present an optimized sample preparation protocol and discuss the challenges of performing solid-state NMR experiments under controlled pH. DMPC/DMPG bilayers show a significant up-field shift and broadening of the main lipid phase transition temperature when lowering the pH from 10.0 to 2.6. Both, strongly acidic and basic pH, cause a significant degree of lipid hydrolysis, which is exacerbated by the presence of PGLa. The characteristic re-alignment of PGLa from a surface-bound to a tilted state is not affected between pH of 7 to 4 in fluid bilayers. On the other hand, in gel-phase bilayers the peptide remains isotropically mobile under acidic conditions, displays various co-existing orientational states at pH7, and adopts an unknown structural state at basic pH.
authors.affiliation: Karlsruhe Institute of Technology (KIT), Institute of Biological Interfaces (IBG-2), POB 3640, 76021 Karlsruhe, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Misiewicz
authors.affiliation: Karlsruhe Institute of Technology (KIT), Institute of Biological Interfaces (IBG-2), POB 3640, 76021 Karlsruhe, Germany.
authors.forename: Sergii
authors.initials: S
authors.lastname: Afonin
authors.affiliation: Karlsruhe Institute of Technology (KIT), Institute of Biological Interfaces (IBG-2), POB 3640, 76021 Karlsruhe, Germany; Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry, Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany. Electronic address: Anne.Ulrich@kit.edu.
authors.forename: Anne S
authors.initials: AS
authors.lastname: Ulrich
mesh_terms: D000595:Amino Acid Sequence; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010715:Phosphatidylglycerols / Q000737:chemistry / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D044366:Transition Temperature
chemical_list: D023181:Antimicrobial Cationic Peptides; D008051:Lipid Bilayers; D008081:Liposomes; D008563:Membrane Lipids; D010715:Phosphatidylglycerols; D010743:Phospholipids; C045866:peptide-Gly-Leu-amide; C002773:dimyristoylphosphatidylglycerol; D004134:Dimyristoylphosphatidylcholine
keywords: Buffering and pH control; Differential scanning calorimetry; Lipid phase transition; Membrane-active peptides; Oriented multilamellar membrane sample; Solid-state (2)H/(31)P/(19)F NMR spectroscopy