Effect of Lipid Composition on the Topography of Membrane-Associated Hydrophobic Helices: Stabilization of Transmembrane Topography by Anionic Lipids

Abstract

To investigate the effect of lipid structure upon the membrane topography of hydrophobic helices, the behavior of hydrophobic peptides was studied in model membrane vesicles. To define topography, fluorescence and fluorescence quenching methods were used to determine the location of a Trp at the center of the hydrophobic sequence. For peptides with cationic residues flanking the hydrophobic sequence, the stability of the transmembrane (TM) configuration (relative to a membrane-bound non-TM state) increased as a function of lipid composition in the order: 1:1 (mol:mol) 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC):1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE) ∼ 6:4 POPC:cholesterol < POPC ∼ dioleoylphosphatidylcholine (DOPC) < dioleoylphosphatidylglycerol (DOPG) ≤ dioleoylphosphatidylserine (DOPS), indicating that the anionic lipids DOPG and DOPS most strongly stabilized the TM configuration. TMstabilization was near-maximal at 20-30mol% anionic lipid, physiologically relevant values. TMstabilization by anionic lipid was observed for hydrophobic sequences with diverse set of sequences (including polyAla), diverse lengths (from 12-22 residues), and various cationic flanking residues (H, R or K), but not when the flanking residues were uncharged. TM-stabilization by anionic lipid was also dependent on the number of cationic residues flanking the hydrophobic sequence, but was still significant with only one cationic residue flanking each end of the peptide. These observations are consistent with TM-stabilizing effects being electrostatic in origin. However, Trp located more deeply in DOPS vesicles relative to DOPG vesicles, and peptides in DOPS vesicles showed increased helix formation relative to DOPG and all other lipid compositions. These observations fit a model in which DOPS anchors flanking residues near the membrane surface more strongly than does DOPG, and/or increases the stability of the TM state to a greater degree than DOPG. We conclude anionic lipids can have significant and headgroup structure-specific effects upon membrane protein topography.

The Effect of Hydrophilic Substitutions and Anionic Lipids upon the Transverse Positioning of the Transmembrane Helix of the ErbB2 (neu) Protein Incorporated into Model Membrane Vesicles

“…In addition, there was a correlation between the extent of transverse shift induced by hydrophilic residues and the previously reported activating effects of these residues upon transformation due to Erb b2 expression 9. Other experiments indicated that anionic lipid could suppress transverse shifts when the juxtamembrane residues flanking the TM sequence were cationic, but not when they had no net charge 21. How such transverse movements and their regulation might influence Erb b2 function is discussed.…”

“…We previously found that anionic lipids strongly stabilize the formation of a TM topography when a hydrophobic sequence is flanked by cationic JM residues 21, 30. This appears to involve Coulombic electrostatic interactions between the cationic JM residues and anionic lipids 21.…”

“…This appears to involve Coulombic electrostatic interactions between the cationic JM residues and anionic lipids 21. Such interactions might also have an important modulatory influence on the tendency of a hydrophobic sequence to shift transverse position in membranes.…”

“…In particular, whether Erb b2 TM helix transverse movement would be suppressed by anionic lipid was investigated using WT sequences and ones with a Gln substitution. The non-ionizable Gln residue was chosen rather than Glu to avoid complications due to effects of anionic lipid effects on anionic residue pKa, and thus anionic residue charge 21.…”

Anionic Lipids Modulate the Activity of the Aquaglyceroporin GlpF

“…Low PG and CL contents initially caused an increased GlpF activity, whereas we observed a decreased GlpF activity at higher negative charge levels. Stabilizing effects of small amounts of anionic lipids on the configuration of TM helices that are flanked by cationic residues were previously described for both PG and PS (50). This effect on GlpF activity, however, seems to be lipid-headgroup specific, as PS did not increase the GlpF activity.…”