Modulating Transmembrane α-Helix Interactions through pH-Sensitive Boundary Residues

Changes in pH can alter the structure and activity. of proteins and may be used by the cell to control molecular function. This coupling can also be used in non-native applications through the design of pH-sensitive biomolecules. For example, the pH (low) insertion peptide (pHLIP) can spontaneously insert into a lipid bilayer when the pH decreases. We have previously shown that the alpha-helicity and helix helix interactions of the TM2 alpha-helix of the proteolipid protein (PLP) are sensitive to the local hydrophobicity at its C-terminus. Given that there is an ionizable residue (Glu-88) at the C-terminus of this transmembrane (TM) segment, we hypothesized that changing the ionization state of this residue through pH may alter the local hydrophobicity of the peptide enough to affect both its secondary structure and helix helix interactions. To examine this phenomenon, we synthesized peptide analogues of the PLP TM2 alpha-helix (wild-type sequence (66)AFQYVIYGTASFFFLYGA-LLLAEGF(90)). Using circular dichroism and Forster resonance energy transfer in the membrane-mimetic detergent sodium dodecyl sulfate, we found that a decrease in pH increases both peptide alpha-helicity and the extent of self-association. This pH dependent effect is due specifically to the presence of Glu-88 at the C-terminus. Additional experiments in which Phe-90 was mutated to residues of varying hydrophobicities indicated that the strength of this effect is dependent on the local hydrophobicity near Glu-88. Our results have implications for the design of TM peptide switches and improve our understanding of how membrane protein structure and activity can be regulated through local molecular environmental changes.