Histidine modulates amyloid-like assembly of peptide nanomaterials and confers enzyme-like activity

Abstract

Amyloid-like assembly is not only associated with pathological events, but also leads to the development of novel nanomaterials with unique properties. Herein, using Fmoc diphenylalanine peptide (Fmoc-F-F) as a minimalistic model, we found that histidine can modulate the assembly behavior of Fmoc-F-F and induce enzyme-like catalysis. Specifically, the presence of histidine rearranges the β structure of Fmoc-F-F to assemble nanofilaments, resulting in the formation of active site to mimic peroxidase-like activity that catalyzes ROS generation. A similar catalytic property is also observed in Aβ assembled filaments, which is correlated with the spatial proximity between intermolecular histidine and F-F. Notably, the assembled Aβ filaments are able to induce cellular ROS elevation and damage neuron cells, providing an insight into the pathological relationship between Aβ aggregation and Alzheimer's disease. These findings highlight the potential of histidine as a modulator in amyloid-like assembly of peptide nanomaterials exerting enzyme-like catalysis.

Fig. 1. Phase change of Fmoc–F–F in His aqueous solution.

a Schematic of phase change of co-assembly Fmoc–F–F by His. b, c SEM and TEM of self-assembled Fmoc–F–F in pure water. Stubby nanorods were obtained. d, e SEM and TEM of co-assembled Fmoc–F–F (His). Thin and long filaments transformed from nanorods were obtained due to the presence of His. f milky white turbid liquid formed by Fmoc–F–F and clear hydrogel formed by Fmoc–F–F (His). g Occurrence of phase transition based on rheological measurements of dynamic frequency sweeps of Fmoc–F–F (His). Three times each experiment was repeated independently with similar results. Representative images are shown. Source data are provided as a Source Data file.

Fig. 2. Cryo-EM characterizations for Fmoc–F–F (His).

a Cryo-EM image of Fmoc–F–F (His) showing primitive spindle filament with heterogeneous diameter. b Two-dimensional classification analysis of Fmoc–F–F (His) showing parallel alignment and entangled bundles in spindle structure. The images correspond to three morphologies (dashed yellow circle, dashed red circle, and dashed blue circle) of boundles in a, respectively. c Negatively stained Cryo-EM image of Fmoc–F–F (His) using uranium acetate confirming the spindle structure of nanofilaments. Three times each experiment was repeated independently with similar results. Representative images are shown.

Fig. 3. Hydrogen bonding interactions of His and Fmoc–F–F destroy π–π stacking of F–F peptides and secondary structure.

a XPS of Fmoc–F–F (His) and Fmoc–F–F. b C 1s peak of Fmoc–F–F (His) from XPS. c ¹H solid nuclear magnetic resonance (NMR) spectra of Fmoc–F–F (His) and Fmoc–F–F. d Percentage of secondary structure (Fmoc–F–F (His)) of amide I region (1600 cm⁻¹ to 1700 cm⁻¹) characterized by FTIR. e Possible interactions in the process of co-assembly between Fmoc–F–F and His. Representative images are shown. Source data are provided as a Source Data file.

Fig. 4. Theoretical analyses of Fmoc–F–F and His co-assembly.

a Fmoc–F–F and His molecules. b Frontier orbitals of π-π stacking interaction of Fmoc–F–F dimer. c Multiple dimers of Fmoc–F–F and His are connected by different hydrogen bonding interaction modes. d Different trimers of 2Fmoc–F–F and His. Fmoc–F–F and 2His are connected by hydrogen bonding interaction modes. The atomic coordinates of the optimized computational models were provided in Supplementary Data 1.

Fig. 5. Enzyme-like activity of Fmoc–F–F (His) and Aβ assembly.

a Peroxidase (POD)-like of Fmoc–F–F (His), Fmoc–F–F, and His, respectively. b Catalase (CAT)-like of Fmoc–F–F (His), Fmoc–F–F, and His, respectively. c POD-like activity of Aβ1–40 and Aβ1–42 filaments. The significant difference was evaluated by a two-tailed unpaired t-test. n = 3 independent samples, bars represent means ± SD, ns means no significance, ****p < 0.0001, **p < 0.01, *p < 0.05). d ESR characterization for •OH generation by POD-like activity of Aβ1–42 aggregates (half a year). e Predicted structures of Aβ1–42 and Aβ1–42 (6His→Ala) by alphafold2. f POD-like of Aβ1–42 and Aβ1–42 (6His→Ala). n = 3 independent samples. Mean ± SD is shown. The significant difference was evaluated by a two-tailed unpaired t-test. **p < 0.01. Representative images are shown. g Cytosolic ROS (cROS) levels of HT-22 cells treated by different concentrations of Aβ1-42 filaments. h Lipid ROS levels of HT-22 cells treated by Aβ1-42 filaments. i Schematic diagram of injecting Aβ1–42 filaments into the hippocampus of SD rats. j Percentage of hippocampal histiocytes with high cROS level in SD rats after treatment of Aβ1–42 filaments. n = 6 biologically independent animals. Mean ± SD is shown. The significant difference was evaluated by a two-tailed unpaired t-test. **p < 0.01, *p < 0.05. Source data are provided as a Source Data file.

Fig. 6. Proposed mechanism of POD-like catalysis of Fmoc–F–F (His) and key structural parameters.

Seven reaction states mediated by POD-like catalysis of Fmoc–F–F (His) was proposed. The whole process can be divided into three major steps: The adsorption of H₂O₂ and (H⁺ + TMB) molecules on Fmoc–F–F (His) (states 1–3); Oxidation reaction of the first TMB molecule oxidized by H₂O₂* under acidic conditions, producing HO*, H₂O*, and oxTMB* (states 4–5); Oxidation of second TMB molecule by second HO* under acidic conditions, producing H₂O* and oxTMB* (states 6–7). The corresponding binding energy of each step can be seen in Supplementary Fig. 33.