Structure And Dynamics Of PH-Responsive Cell-Penetrating Peptides At Biological Membrane Revealed By Sum Frequency Generation Vibrational Spectroscopy

pH-sensitive cell penetrating peptides have broad prospects in biomedical application and biophysical basic research. Such as targeted drug translocation and targeted diagnosis and therapy for disease, as a model for studying protein folding and membrane fusion mechanisms. The main feature of this kind of peptide is that it can sense environmental pH change and transform from random coil structure to ?-helical structure below a critical pH value. The loop-helix transition is a critical step in the transmembrane process, while previous studies lack the effective technique of characterizing loop to ?-helical structure transition and mainly focus on the structure of them in solution. In this paper, two representative peptides, GALA and pHLIP,were used as the model to systematically investigate pH response of them in different lipid bilayers by combing second-ordered amide I and amide III spectral signals,which can accurately differentiate the loop and a-helical structures at the interface. It is found that GALA can insert into fluid-phase lipid bilayers even at neutral pH, while lies down on the gel-phase lipid bilayer surface. In acidic conditions, GALA inserts into both fluid-phase and gel-phase lipid bilayers. GALA adopts a mixed loop and a-helical structures in lipid bilayers. Besides, the reversible activation of GALA in lipid bilayers depends on the nature of lipid. After membrane insertion, GALA exits from the negative phosphoglycerol and positive ethylphosphocholine lipid bilayers at neutral pH. But it does not exit from the zwitterionic phosphocholine lipid bilayers.pHLIP can form ?-helical structures on the surface of or insert into zwitterionic phosphocholine lipid bilayers and positive ethylphosphocholine lipid bilayers at neutral pH while cannot interact with negative phosphoglycerol lipid bilayers. When the environment becomes acidic, pHLIP structural response is not sensitive and there is no significant signals of ?-helical structures and interactions in negative phosphoglycerol lipid bilayers. Our results provide a high resolution molecular structural information and indicate that the structure and kinetics of the two peptides are related to the physical properties of the phospholipid membrane, which also provides a way to regulate the structure of the peptides through changes in lipid composition. However, the structure and dynamics of this two peptides at interface are different from that in solution, which also enriches our understanding of these peptides, aiding in understanding the mechanism of interaction between peptide and membrane and improving safety and efficiency of this kind of peptide.