Position and Ionization State of Asp in the Core of Membrane-Inserted α Helices Control Both the Equilibrium between Transmembrane and Nontransmembrane Helix Topography and Transmembrane Helix Positioning

Abstract

The behavior of model-membrane-inserted polyLeu-rich peptides containing Asp residues located at various positions in their hydrophobic core was investigated. The topography of the bilayer-inserted alpha helices formed by these peptides was evaluated by measuring the emission lambda(max) and quenching the fluorescence of a Trp at the center of the peptide sequence. When Asp residues were protonated (at low pH), peptides that were incorporated into vesicles composed of dioleoylphosphatidylcholine (DOPC) adopted a topography in which the polyLeu sequence predominantly formed a normal transmembrane (TM) helix. When Asp residues were ionized (at neutral or high pH), topography was altered in a manner that would allow the charged Asp residues to reside near the bilayer surface. In DOPC vesicles, most peptides repositioned so that the longest segment of consecutive hydrophobic residues (12 residue minimum) formed a truncated/shifted TM structure. However, peptides with one or two charged Asp residues close to the center of the hydrophobic sequence and thus lacking even a 12-residue continuous hydrophobic segment, formed a helical non-TM state locating near the bilayer surface. At low pH, incorporation of the peptides into thicker bilayers composed of dierucoylphosphatidylcholine (DEuPC) resulted in the formation of a mixture of the normal TM state and the non-TM helical state located near the bilayer surface. In DEuPC vesicles at high pH, the non-TM state tended to predominate. How Asp-ionization-dependent shifts in helix topography may regulate the function of membrane proteins exposed to environments with differing pH in vivo (e.g., endosomes) is discussed.

Buried lysine, but not arginine, titrates and alters transmembrane helix tilt

“…We note also that a pK a of about 8.7 has been suggested for the Asp residue in the transmembrane helix formed by the peptide K 2 GL 7 DLWL 9 K 2 A (30). Although the dielectric gradient through the bilayer is very steep, such that pK a values will vary substantially with distance from the bilayer center (11), it is conspicuous that the reported values for Asp (30) and Lys (this work) are both shifted by about 4 pH units in favor of the neutral form in the bilayer compared with aqueous solution. Because K12 in GWALP23 is located closer to the bilayer center in a lower dielectric environment than K14, one expects an even lower pK a for K12 than for K14.…”

Juxtamembranous aspartic acid in Insig-1 and Insig-2 is required for cholesterol homeostasis

“…The DRSR sequences in Insig proteins and SREBPs may dictate the conformation of the protein at the surface of the membrane bilayers. The aspartic acid in the tetrapeptide may bind to the positively charged head groups of phospholipids to stabilize the correct folding of the transmembrane helix (19). Although the residues surrounding Asp-205 (FDRSR) are conserved in all Insig proteins, the aspartic acid residue appears to be the only one that is essential for function.…”

Endoplasmic Reticulum Retention and Associated Degradation of a GABAA Receptor Epilepsy Mutation That Inserts an Aspartate in the M3 Transmembrane Segment of the α1 Subunit

“…Could a single point mutation cause misfolding of the majority of translated ␣1 protein? It has been shown in model peptides that at neutral or high pH, a single aspartate positioned near the center of a transmembrane helix would disrupt the helix to allow the aspartate to reside near the aqueous surface (37). Therefore, at physiological pH, it may be expected that a substantial number of ␣1(A322D) M3 segments would fail to form stable transmembrane helices after translation.…”