Characterization of an Intermediate for the Formation of the Transmembrane Helix of pHLIP Peptide

Abstract

The pHLIP® -pH (low) insertion peptide- binds to the surface of lipid bilayers as a disordered peptide at neutral pH. However, when the pH is lowered, pHLIP inserts across the membrane to form a transmembrane (TM) helix. Peptide insertion is reversed when the pH is raised above the characteristic pKa, which is 6.0 for POPC liposomes. Due to its unique properties pHLIP serves as a good model system to study the process of folding/insertion and exit/unfolding of a peptide into/from a membrane. We previously found that the process of insertion of pHLIP in POPC phospholipid bilayer occurs in several steps with different intermediates (Andreev et al 2010, PNAS). The number of intermediates correlates with the number of protonatable groups in pHLIP's sequence. For pHLIP, the pH-dependent insertion and folding processes are coupled, and single sigmoidal transition is observed. Interestingly, we have found that for two pHLIP variants (D14A and D25A where each aspartic residue in TM part is replaced by alanine), the pH curve shows two independent sigmoidal transitions, suggesting that the mutations stabilize an intermediate state along the membrane-attached to transmembrane pathway. We implemented circular dichrosism (CD), oriented CD, fluorescence, FRET to elucidate the nature of these intermediates. From our experiments we inferred that the intermediates correspond to a quasi-transmembrane state. We hypothesize that the intermediate emerges from the protonation of acidic residues at the peptide C-terminal end, and complete TM folding happens with the protonation of the aspartic acid residue located in the hydrophobic core of the membrane. These results further our understanding of formation of transmembrane helix during spontaneous process of insertion of a polypeptide in membrane.