Peptide Stapling: A Strategy for Enhanced Molecular Recognition of C-Reactive Protein

This is the authors' abstract. We don't add key points for this paper.

In our previous studies, we identified three linear CRP‑binding peptides (P2, P3, and P9), which were subsequently characterized using experimental approaches and in silico analysis. The P3 peptide was successfully used as a sensing element in a microfluidic sensing device for CRP recognition 1. However, linear peptides have some limitations, such as conformational instability. Thus, in our study, we constrained the peptide structure using a stapling approach (P1) to address this problem. In silico analysis showed that only the P9 peptide retained CRP-binding activity after the stapling reaction among other CRP-binding peptides reported in our previous studies 1. Calculations indicated that the staple-bound peptide P1 has a higher affinity for CRP than the linear peptide P9, which lacks a staple. Furthermore, the calculated binding energy for P1 was lower than that obtained for the stapled peptides P2 and P3. The staple in peptide P1 was formed via ring-closing olefin metathesis (RCM). Structural differences between the new stapled P1 peptide and the linear P9 peptide were demonstrated using transmission electron microscopy (TEM) and circular dichroism (CD) measurements. The stapled P1 peptide was applied to recognize CRP on silicate-modified indium tin oxide-coated glass electrodes. The electrodes obtained with P1 exhibit a greater affinity for CRP, even in the presence of proteolytic enzymes, than those modified with the linear P9 peptide. The thermal and proteolytic stability of the peptides was evaluated using electrochemical analyses and CD.