pH-induced insertion of pHLIP into a lipid bilayer: In-situ SEIRAS characterization of a folding intermediate at neutral pH

The pH low insertion peptide (pHLIP) is a pH-sensitive cell penetrating peptide that transforms from an unstructured coil on the membrane surface at pH > 7, to a transmembrane (TM) alpha-helix at pH < 5. By exploiting this unique property, pHLIP attracts interest as a potential tool for drug delivery and visualisation of acidic tissues produced by various maladies such as cancer, inflammation, hypoxia etc. Even though the structures of initial and end states of pHLIP insertion have been widely accepted, the intermediate structures in between these two states are less clear. Here, we have applied in situ Surface-Enhanced Infrared Absorption spectroscopy to examine the pH-induced insertion and folding processes of pHLIP into a solid-supported lipid bilayer. We show that formation of partially helical structure already takes place at pH only slightly below 7.0, but with the helical axis parallel to the membrane surface. The peptide starts to reorientate its helix from horizontal to vertical direction, accompanied by the insertion into the TM region at pH < 6.2. Further insertion into the TM region of the peptide results in an increase of inherent alpha-helical structure and complete secondary structure formation at pH 5.3. Analysis of the changes of the carboxylate vibrational bands upon pH titration shows two distinctive groups of aspartates and glutamates with pK(a) values of 4.5 and 6.3, respectively. Comparison to the amide bands of the peptide backbone suggests that the latter Asp/Glu groups are directly involved in the conformational changes of pHLIP in the respective intermediate states.

Automated summary

The pH low insertion peptide (pHLIP) is a pH-sensitive cell penetrating peptide that undergoes conformational changes from a coil to an alpha-helix in response to pH. It has potential applications in drug delivery and visualization of acidic tissues. In this study, the pH-induced insertion and folding processes of pHLIP into a lipid bilayer were examined using surface-enhanced infrared absorption spectroscopy. The results showed that the peptide forms a partially helical structure at slightly acidic pH and undergoes further reorientation and insertion into the membrane at lower pH. The formation of alpha-helical structure and complete secondary structure occurs at pH 5.3. Analysis of the vibrational bands suggests the involvement of specific amino acid groups in the conformational changes of pHLIP in different pH states.