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 sequence of the transmembrane (TM) helix of Erb b2, a member of the epidermal growth factor receptor family, can influence its activity. In this report, the sequence and lipid dependence of the transverse position of a model membrane-inserted peptide containing the Erb b2 TM helix and juxtamembrane (JM) residues was studied. For the Erb b2 TM helix inserted into phosphatidylcholine vesicles, the activating V664E mutation was found to induce a transverse shift involving the movement of the E residue towards the membrane surface. This shortened the effective length of the TM spanning portion of the sequence. The transverse shift was observed with both the E664 residue in the uncharged and charged state, but the extent of the shift was larger when the E residue was charged. When a series of hydrophilic residues was substituted for V664 the resulting transverse shifts at pH 7 decreased in the order D,H>E>Q>K>G>V. Except for His, this order is strongly correlated to that reported for the degree to which these substitutions induce cellular transformation when introduced into full length Erb b2. To examine the effect of lipid on transverse shift, the uncharged V664Q mutation was studied. The presence of 20% of the anionic lipid dioleoylphosphatidylserine (DOPS) in the model membrane vesicles, which introduces a physiologically relevant level of anionic lipid, did not affect the degree of transverse shift. However, in the case of a peptide containing a V674Q substitution, in which the Q is closer to the C-terminal of the Erb b2 TM helix than the N-terminal, transverse shift was suppressed in vesicles containing 20% DOPS. This suggests that the interaction of the cationic JM residues flanking the C-terminus of the Erb b2 TM helix interact with anionic lipids to anchor the C-terminal end of the TM helix. This anchoring site may act as a pivot which amplifies transverse movements of the Erb b2 TM segment to induce a large swinging-type motion in the extracellular domain of the protein, affecting Erb b2 activity. Interactions interrupting C-terminal JM residue association with anionic lipid might partly impact Erb b2 activity by disrupting this pivoting.

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Receptor Tyrosine Kinases (RTKs) play key roles in cell growth, differentiation, metabolism, and migration. These single-pass receptors conduct biochemical signals by dimerizing in the plasma membrane. The process of lateral dimerization, which controls the distribution between inactive monomers and active dimers, serves as a key regulator of the biochemical processes that determine cell fate. Enhanced dimerization leads to persistent autocrine activation and tumorigenesis, or impaired growth. An understanding of the dimerization process as a function of interaction energies, protein concentration and ligand concentration, is lacking. Our laboratory is developing methodologies that yield quantitative information about RTK dimerization and activation in cellular membranes. These methods will enable biomedical researchers to study the quantitative aspects of signal transduction in the context of the biological membrane.