Kanazawa University research: Frequency modulated AFM sheds light on how dipeptides help organize, immobilize and catalyze

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Mar 04, 2025, 07:15 ET

KANAZAWA, Japan, March 4, 2025 /PRNewswire/ -- Researchers at Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, observe the configuration of different dipeptides on graphite electrodes and the subsequent arrangement of catalytic hemin on them to get an idea of the factors affecting its catalytic activity.

Self-assembled peptides have shown great promise for immobilizing and exploiting enzymes in catalytic applications. However, so far little has been known as to the structures of these self-assembled peptides and how this might affect the function of the enzyme immobilized. Now researchers led by Ayhan Yurtsever and Takeshi Fukuma at Kanazawa University, WPI-NanoLSI and Marie Sugiyama and Yuhei Hayamizu at Institute of Science Tokyo have compared the morphology and activity of hemin adsorbed on different dipeptide nanostructures using atomic force microscopy (AFM), cyclic voltammetry and H₂O₂ reduction reactions to see which offers the best performance and why (Figure 1).

The researchers used frequency modulated atomic force microscopy to study the structures that self-assembled from droplets of (XH)₄ peptide solution on a graphite electrode, where H is histidine and X is an amino acid – either Y, L or V. Their observations indicated that dipeptides self-assemble into repeating nanostructures resembling 2D crystals, with (YH)4 exhibiting the most ordered and stable configuration (Figure 2).

They then replaced the droplet of peptide solution with a droplet of hemin solution and used AFM to observe the configuration of the hemin as it bound to the peptide structure. They found the hemin aggregated on the dipeptide structures, and further observations with high-speed AFM revealed that the hemin formed wires as well as aggregates, and that while the wires were stationary the aggregates seemed to hop along and between rows of the dipeptide (Figure 3).

The researchers used cyclic voltammetry to measure how densely hemin bound to the dipeptide structures and found that it bound most densely to (YH)₄. They attribute this to the tyrosine in (YH)₄, which interacts with porphyrin through π-π interactions. However, adding just porphyrin to the dipeptide bound hemin structures had little effect, from which they deduced that "the Fe atom in hemin is critical for its interaction with peptides, and that the binding is not solely driven by π−π stacking interactions," as they report in ACS Nano. While the density of hemin binding to (LH)₄ was close to that for (VH)₄, they found it bound slightly more densely to (VH)₄, which they attribute to the greater hydrophobicity.

On applying a reduction current to the electrodes, the iron in hemin is reduced to the ferrous (+2) oxidation state. It can then reduce H₂O₂, thereby recovering its ferric oxidation state. Comparisons of how quickly the hemin bound structures reduce H₂O₂ revealed that hemin bound to (YH)₄ had the highest catalytic activity, although this is unlikely due to the greater density of hemin at this surface since the densities for all three dipeptides were all within the same order of magnitude. Instead, the researchers suggest the greater reducing power of hemin bound to (YH)₄ is on account of the more stable scaffold offered by that dipeptide (Figure 4).

"This research highlights the potential of simple peptide designs to create artificial enzymes with robust and durable catalytic interfaces for electrochemical applications," conclude the researchers in their report. "Furthermore, the peptides' ability to self-assemble on two-dimensional materials makes them promising candidates for biosensing applications."
Glossary

Atomic force microscopy

This imaging technique uses a nanosized tip at the end of a cantilever that is scanned over a sample. It can be used to determine the topography of a sample surface from the change in the strength of forces between the tip and the sample with distance, and the resulting deflection of the cantilever. It was first developed in the 1980s but a number of modifications have augmented the functionality of the technique since. It is better suited to imaging biological samples than the scanning tunnelling microscope developed that had been developed because it does not require a conducting sample.

In the 2000s Toshio Ando at Kanazawa University was able to improve the scanning speed to such an extent that moving images could be captured. This allowed people to use the technique to visualize molecular processes for the first time.

Catalysts

Catalysts influence the rate of a reaction without actually being used up in the reaction themselves. They can speed up the rate of all kinds of reactions, including the redox chemistry in the reduction of H₂O₂, and are prized in industry for improving yield and the profitability of processes.

Redox chemistry describes a host of reactions that involve the gaining (reduction) and losing (oxidation) of electrons by ions in the reaction. It sometimes manifests as the gaining of hydrogen (reduction) or oxygen (oxidation). The generation of water and oxygen from H₂O₂ is an example of a redox reaction where H₂O₂ is reduced to H₂O and O₂.

Porphyrin

Porphyrin is an organic compound made up of a ring of four substituted "pyrrole" ring molecules strung together with methine bridges (=CH-). Pyrrole is a cyclic molecule with the formula C₄H₄NH but in porphyrin other groups may be substituted in. An important porphyrin for living organisms is heme, which carries oxygen in the blood. Chlorophyl is also a porphyrin derivative

Hemin

Hemin is an iron-containing porphyrin found in the blood. It is the catalytic centre for many different proteins including cytochromes, peroxidases, myoglobins and hemoglobin. The iron in hemin is ferric, that is, it is in the +3 oxidation state (Fe³⁺).

π−π interactions

In aromatic molecules atoms are bound in the ring by π bonds, a type of covalent bond that takes its name from the shape of the electron orbital which forms lobes on either side of the atoms. π−π stacking describes the non-covalent interactions when these rings stack on each other.

Reference

Marie Sugiyama, Ayhan Yurtsever, Nina Uenodan, Yuta Nabae, Takeshi Fukuma, and Yuhei Hayamizu Hierarchical Assembly of Hemin-Peptide Catalytic Systems on Graphite Surfaces ACS NANO 2025.

DOI： 10.1021/acsnano.4c15373

URL: https://pubs.acs.org/doi/full/10.1021/acsnano.4c15373

Funding acknowledgements

Y.H. acknowledges support from the Precise Measurement Technology Promotion Foundation (PMTP-F), JSPS KAKENHI Grants 20H02564, 20H03593, 22H05408 and 24H01124, and JST CREST Grant Number JPMJCR24A4, Japan. T.F. acknowledges support from the World Premier International Research Center Initiative (WPI), MEXT, Japan, and JSPS KAKENHI Grant Number 21H05251.

Fig: https://nanolsi.kanazawa-u.ac.jp/wp/wp-content/uploads/Eye-catching-image_ACS-Nano_2025.2.jpg

Caption Peptide self-assembly and subsequent hemin adsorption on graphite substrate. (A) High-resolution AFM image showing the molecular arrangement of (YH)4 peptides, forming 2D crystalline lattices on graphite in water. (B) Initial stage of hemin binding on self-assembled (YH)4 peptide nanostructures, revealing the formation of relatively unstable molecular rows along peptide lattices. (C) At later stages of adsorption, the hemin molecules form more stable and densely packed rows that ultimately cover the underlying peptide lattices completely.

https://nanolsi.kanazawa-u.ac.jp/wp/wp-content/uploads/Figure-1_ACS-Nano_2025.2.jpg
Fig. 1 A schematic illustration depicting the catalytic reaction mechanism of the system.

https://nanolsi.kanazawa-u.ac.jp/wp/wp-content/uploads/Figure-2_ACS-Nano_2025.2.jpg
Fig. 2: In situ FM-AFM images showing the unit cells of each peptide assemblies on graphite.

https://nanolsi.kanazawa-u.ac.jp/wp/wp-content/uploads/Figure-3_ACS-Nano_2025.2.jpg
Fig 3: In situ AFM image showing the immobilization of hemin on self-assembled (YH)₄ peptides, revealing the formation of hemin molecular rows along peptide lattices.

https://nanolsi.kanazawa-u.ac.jp/wp/wp-content/uploads/Figure-4_ACS-Nano_2025.2.jpg
Fig. 4: Current density at −0.8 V as a function of H₂O₂ concentration for each peptide, with fitting curves shown as red solid lines. I_max represent the maximal current density.

Contact

Kimie Nishimura (Ms)
Project Planning and Outreach, NanoLSI Administration Office
Nano Life Science Institute, Kanazawa University
Email: [email protected]

Kakuma-machi, Kanazawa 920-1192, Japan

About Nano Life Science Institute (WPI-NanoLSI), Kanazawa University

Understanding nanoscale mechanisms of life phenomena by exploring "uncharted nano-realms".

Cells are the basic units of almost all life forms. We are developing nanoprobe technologies that allow direct imaging, analysis, and manipulation of the behavior and dynamics of important macromolecules in living organisms, such as proteins and nucleic acids, at the surface and interior of cells. We aim at acquiring a fundamental understanding of the various life phenomena at the nanoscale.

https://nanolsi.kanazawa-u.ac.jp/en/

About the World Premier International Research Center Initiative (WPI)

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About Kanazawa University

As the leading comprehensive university on the Sea of Japan coast, Kanazawa University has contributed greatly to higher education and academic research in Japan since it was founded in 1949. The University has three colleges and 17 schools offering courses in subjects that include medicine, computer engineering, and humanities.

The University is located on the coast of the Sea of Japan in Kanazawa – a city rich in history and culture. The city of Kanazawa has a highly respected intellectual profile since the time of the fiefdom (1598-1867). Kanazawa University is divided into two main campuses: Kakuma and Takaramachi for its approximately 10,200 students including 600 from overseas.
http://www.kanazawa-u.ac.jp/en/

SOURCE Kanazawa University