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.

Membrane Thinning and Thickening Induced by Membrane-Active Amphipathic Peptides

“…Furthermore, both methods are compatible with oriented bilayer samples, which were used for three reasons. Firstly, this way the results of the present study can be directly related with our previous solid-state NMR measurements of the orientation of the selected peptides in mechanically oriented membranes (Salgado et al, 2001; Glaser et al, 2005; Grage et al, 2006; Tremouilhac et al, 2006; Afonin et al, 2008a,b, 2014; Strandberg et al, 2013; Wadhwani et al, 2014; Misiewicz et al, 2015a,b). Secondly, hydrated oriented samples provide a well-defined bilayer structure and morphology, with a uniform environment for the peptides.…”

“…As two prototypic peptides of this peptide super-family, we studied the membrane interaction of PGLa and magainin 2 (Mag2) from the skin of X. laevis . PGLa is known to adopt three discrete alignment states, depending largely on peptide:lipid ratio, but also on temperature, pH, and lipid composition (Glaser et al, 2005; Afonin et al, 2008a, 2014; Misiewicz et al, 2015b). At low concentration, PGLa assumes a surface-bound state (S-state) with the helix axis parallel to the bilayer plane, but it gets tilted into an oblique alignment above a peptide:lipid threshold of ~1:80 (T-state).…”

Recent Solid-State NMR Studies of Membrane-Bound Peptides and Proteins

“…The characteristic realignment of PGLa from a surface-bound to a tilted state in the fluid bilayer was not affected between pH 7 and 4. On the other hand, in gel-phase bilayers, the peptide was shown to remain isotropically mobile under acidic conditions, to display various coexisting orientation states at pH 7, and to adopt an unknown structural state at basic pH [59].…”

“…The lipid and peptide components were characterized independently, using sample with same orientation under typical conditions of maximum hydration. It showed the importance of an optimized sample preparation protocol of performing solid-state NMR experiments under controlled pH [59]. DMPC/DMPG bilayers showed a significant up-field shift and broadening of the main lipid-phase-transition temperature when the pH was lowered from 10.0 to 2.6.…”

“…Solid-state NMR is particularly well suited for elucidating the dynamics, topology, orientation, and high-resolution structures of peptides in the bilayer environment using model and cell membranes. Peptides analyzed using this approach include venoms (melittin [27,29], bombolitin [54]), antimicrobial peptides (lactoferrampin [55], lactoferricin [56], PGLa [57][58][59], MSI-78 [60][61][62], LL-37 [63], pardaxin [62,64], magainin [65], P5 peptide [66], PG-1 [67], piscidin [68], crown ether containing 14-mer peptide [69,70]), K + channels (Shaker B ball peptide [71]), Neu receptor peptide [72], antibiotic peptides (alamethicin [73][74][75]), opioid peptides (dynorphin [76]), and κ-opioid receptor fragment (ECL-II [77]). In this section, the structure details of some of these peptides are reviewed in detail.…”