Optimizing the detection of pH-dependent conformational changes in pH-low insertion peptides using microfluidic modulation spectroscopy (MMS)

Introduction: The acidic microenvironment of pancreatic ductal adenocarcinoma (PDAC) can be targeted by pH-responsive peptides that undergo conformational changes upon protonation of their negatively charged residues. While microfluidic modulation spectroscopy (MMS) has been successfully used to detect higher-order structure in large proteins, there is room for optimization in the reliable detection of secondary structure for small peptides (< 30 amino acids). We utilized two pH-responsive peptides, V7 and V3, and an inactive peptide, K7, to investigate the effects of buffer conditions and the presence of liposomes on the ability of MMS to detect pH-based conformational changes in these small peptides. Methods: Peptides were synthesized using the CEM LibertyBlue microwave technology. 10 mM and 20 mM phosphate buffer solutions (PBS) were prepared at pH 7.4 and 6.6. Liposomes were prepared using the thin film hydration technique, and then characterized with dynamic light scattering (DLS) to find size, polydispersity index, and surface potential. The RedShiftBio Aurora MMS system was used to analyze the secondary structure of V7, V3, and K7 at either pH 7.4 or 6.6 PBS with liposomes or without liposomes. Each buffer solution was prepared with 1 mg/mL of peptide and sonicated for 30 minutes before MMS reading. RedShiftBio higher-order structure and Gaussian analysis software were used to compare conformational changes between pH 7.4 to 6.6 for each peptide in the various buffer conditions. Results: DLS revealed the liposomes to be 122 nm with a polydispersity index of 0.207 and a surface potential of +32.9 mV. The expected conformational changes for V7 and V3 based on pH could not be adequately detected without the presence of liposomes in the solution. 10 mM PBS with a concentration of 10 6 liposomes per mL was the optimal solution for characterizing the peptides, with V7 and V3 experiencing an increase in alpha-fold at pH 6.6 compared to 7.4, and K7 experiencing either no change or less alpha-fold at pH 6.6 compared to 7.4. Conclusion: A low salt concentration solution containing liposomes was the ideal environment for the peptides V7 and V3 to undergo a pH-dependent conformational change that could be detected by MMS. MMS is a relatively quick method for determining peptide secondary structure with a low limit of quantification. Funding: R01CA2810190