In‐Depth Structural Analysis of a Stapled Pentapeptide and Its Assembly Into Straight α‐Helices

Abstract

Peptide self‐assembly is a complex hierarchical process involving the progressive formation of secondary structures, such as α‐helices, β‐sheets, and turns, during the early stages. It is precisely these multi‐component building blocks that contribute to the complexity of protein assemblies in living organisms. While coiled coils are well‐understood in protein folding, determining the structural characteristics governing their lateral packing remains challenging. Here, a stapled pentapeptide (CIHs) that forms straight α‐helices are reported. Using single crystal X‐ray diffraction and Microcrystal electron diffraction (Micro‐ED), the atomic‐level assembly mechanism of CIHs is investigated. This study describes the specific geometric standards based on these α‐helical building blocks and their interactions. By modulating the hydrophobic interface between these blocks via side‐chain alterations, this study validates that these mutant assemblies can inherit the specific spatial geometry and regular interaction interfaces of straight α‐helices. These results provide a simple template for exploring the hierarchical assembly of straight α‐helices and studying the impact of side chains on lateral packing, opening new avenues for the development of high‐order peptide assemblies based on straight α‐helices.