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Cell membranes are responsible for a range of biological processes that require interactions between lipids and proteins. While the effects of lipids on proteins are becoming better understood, our knowledge of how protein conformational changes influence membrane dynamics remains rudimentary. Here, we performed experiments and computer simulations to study the dynamic response of a lipid membrane to changes in the conformational state of pH-low insertion peptide (pHLIP), which transitions from a surface-associated (SA) state at neutral or basic pH to a transmembrane (TM) α-helix under acidic conditions. Our results show that TM-pHLIP significantly slows down membrane thickness fluctuations due to an increase in effective membrane viscosity. Our findings suggest a possible membrane regulatory mechanism, where the TM helix affects lipid chain conformations, and subsequently alters membrane fluctuations and viscosity. 

The pH-low insertion peptide (pHLIP) is an anionic membrane-active peptide with promising potential for applications in imaging of cancer tumors and targeted delivery of chemotherapeutics. The key advantage of pHLIP lies in its acid sensitivity: in acidic cellular environments, pHLIP can insert unidirectionally into the plasma membrane. Partitioning–folding coupling is triggered by titration of the acidic residues in pHLIP, transforming pHLIP from a hydrophilic to a hydrophobic peptide. Despite this knowledge, the reverse pathway that leads to exit of the peptide from the plasma membrane is poorly understood. Our hypothesis is that sequential deprotonation of pHLIP is a prerequisite for exit of the peptide from the plasma membrane. We carried out molecular dynamics (MD) simulations to characterize the effect that deprotonation of the acidic residues of pHLIP has on the stability of the peptide when inserted into a model lipid bilayer of 1-palmitoyl-2-oleoyl-sn-3-phosphocholine (POPC). Initiation of the exit mechanism is facilitated by a complex relationship between the peptide, bulk solvent, and the membrane environment. As the N-terminal acidic residues of pHLIP are deprotonated, localized loss of helicity drives unfolding of the peptide and more pronounced interactions with the bilayer at the lipid–water interface. Deprotonation of the C-terminal acidic residues (D25, D31, D33, and E34) leads to further loss of secondary structure distal from the C-terminus, as well as formation of a water channel that stabilizes the orientation of pHLIP parallel to the membrane normal. Together, these results help explain how stabilization of intermediates between the surface-bound and inserted states of pHLIP occur and provide insights into rational design of pHLIP variants with modified abilities of insertion. 

G protein-coupled receptors (GPCRs) have long been shown to exist as oligomers with functional properties distinct from those of the monomeric counterparts, but the driving factors of oligomerization remain relatively unexplored. Herein, we focus on the human adenosine A 2A receptor (A 2A R), a model GPCR that forms oligomers both in vitro and in vivo. Combining experimental and computational approaches, we discover that the intrinsically disordered C-terminus of A 2A R drives receptor homo-oligomerization. The formation of A 2A R oligomers declines progressively with the shortening of the C-terminus. Multiple interaction types are responsible for A 2A R oligomerization, including disulfide linkages, hydrogen bonds, electrostatic interactions, and hydrophobic interactions. These interactions are enhanced by depletion interactions, giving rise to a tunable network of bonds that allow A 2A R oligomers to adopt multiple interfaces. This study uncovers the disordered C-terminus as a prominent driving factor for the oligomerization of a GPCR, offering important insight into the effect of C-terminus modification on receptor oligomerization of A 2A R and other GPCRs reconstituted in vitro for biophysical studies.