Structural Analyses of Designed -Helix and -Sheet Peptide Nanofibers Using Solid-State Nuclear Magnetic Resonance and Cryo-Electron Microscopy and Introduction of Structure-Based Metal-Responsive Properties

The 21-residue peptide alpha 3, which is artificially designed and consists of three repeats of 7 residues, is known to rapidly assemble into the alpha-helix nanofiber. However, its molecular structure within the fiber has not yet been fully elucidated. Thus, we conducted a thorough investigation of the fiber's molecular structure using solid-state NMR and other techniques. The molecules were found to be primarily composed of the alpha-helix structure, with some regions near the C- and N-terminal adopting a 3(10)-helix structure. Furthermore, it was discovered that beta-sheet hydrogen bonds were formed between the molecules at both ends. These intermolecular interactions caused the molecules to assemble parallelly in the same direction, forming helical fibers. In contrast, we designed two molecules, CaRP2 and beta KE, that can form beta-sheet intermolecular hydrogen bonds using the entire molecule instead of just the ends. Cryo-EM and other measurements confirmed that the nanofibers formed in a cross beta structure, albeit at a slow rate, with the formation times ranging from 1 to 42 days. To create peptide nanofibers that instantaneously respond to changes in the external environment, we designed several molecules (HDM1-3) based on alpha 3 by introducing metal-binding sites. One of these molecules was found to be highly responsive to the addition of metal ions, inducing alpha-helix formation and simultaneously assembling into nanofibers. The nanofibers lost their structure upon removal of the metal ion. The change occurred promptly and was reversible, demonstrating that the intended level of responsiveness was attained.