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

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.