De Novo Design of a Nanopore for Single-Molecule Detection that Incorporates a β-Hairpin Peptide

Like Comment
Read the paper

To begin

Thank you very much for accepting my paper in Nature Nanotechnology. We would like to thank everyone involved in this journal. I would also like to express my deepest appreciation to Prof. Kawano for his guidance, to the members of Konan University and Yokohama National University for their cooperation as collaborators, and to the students at Tokyo University of Agriculture and Technology for their support in this research.

 

Inspiration for this study

This research is the result of investigations conducted during my master’s course. I am particularly interested in the advanced functions of proteins. Natural proteins are composed of only 20 amino acids, and yet they have complex and sophisticated functions. The functional mechanisms of proteins have been gradually elucidated to the extent that in recent years, it has become possible to create proteins artificially. At the same time there has been an increase in the number of researchers who think, “If I can create a protein with the desired function, it will be useful in a variety of fields.”

The first paper I read was “Lear, J. D., Wasserman, Z. R. & DeGrado, W. F. Science 240, 1177-1181 (1988)”. This paper described the artificial design of peptides, which are short proteins. The artificial peptide consisted of only two amino acids, but it had the ability to assemble in lipid membranes to form channels. The contrast between the simplicity of the sequence and the sophistication of the function inspired me to design a similar artificial peptide. Compared with many researchers who have similar ideas, I was lucky that I belonged to the Kawano laboratory. The laboratory here has a system that allows us to evaluate a single nanopore, which is a nano-sized hole formed by membrane proteins or peptides in lipid membranes.

I then wondered why most of the structures of artificially-designed peptide nanopores to date were α-helices. Most of the proteins used in nanopore sensing, such as α-hemolysin and MSPA, have a β-barrel as the transmembrane structure. I realized that if we can design nanopore-forming peptides with a β-sheet structure, which has not been previously demonstrated, we may be able to create nanopores which are more suitable for sensing than the conventional α-helix. Therefore, I attempted to design a β-sheet peptide that forms nanopores in lipid membranes.

 

Difficulty in this research and the importance of collaboration

We faced many difficulties in this research that we did not initially anticipate. To overcome these difficulties, it was a great help to combine the technologies of various fields and partners.

The initial and biggest challenge occurred in the first two months. Until I started the research, I was unaware that transmembrane beta-sheets are very difficult to synthesize using common methods, because they are hydrophobic and readily aggregate. Therefore, we sought help from the Usui Laboratory of Konan University. The Usui Laboratory has advanced synthesis technology and has succeeded in synthesizing β-sheet peptides by using special dipeptides. After consulting with them, they synthesized for us some peptides with a sequence modification and radiation isotopes, and we are very grateful for their help.

Another major difficulty was the acquisition of structural data of the synthesized nanopores. It is difficult to clarify the detailed structure of nanopores in lipid membranes from electrophysiological data alone. So we could not comfortably answer the question, “Are the nanopores really forming beta-sheet structures?” asked at academic conferences. Therefore, we asked the Kawamura Laboratory of Yokohama National University for assistance in acquiring structural data. Using the technology of the Kawamura Laboratory, the nanopore structure in lipid membranes was clarified by molecular dynamics simulations. Furthermore, they succeeded in obtaining solid state NMR experimental data to support the elucidated nanopore structure.

In this way, it is clear that joint research has great power in overcoming difficulties. The achievements in this paper would not have been possible without the combined knowledge and skills of the three laboratories. We would like to thank all of our collaborators for their cooperation in this research.

 

What I find interesting about research

I think one of the most interesting aspects of scientific research is examining the causes of unexpected events and taking a problem-solving approach to investigate them. In this research, we designed peptides that formed nanopores and detected DNAs and proteins. Channel current measurement is a powerful tool, allowing us to indirectly observe the invisible phenomenon of molecules passing through the nanopores by using changes in current values. However, when current measurements are repeated, sometimes unexpected current signals are observed, and conversely, sometimes no current signals are observed. For example, when we first started the experiment of passing DNA through the nanopore, we could not observe the translocation events even after adding DNA. We made the following hypotheses:

  • The experimental conditions such as DNA concentration and applied voltage were not appropriate.
  • The size of the nanopore was too small and the DNA did not pass through.
  • The DNA passed through the nanopore, but too quickly to detect.

After investigation, we succeeded in detecting the passing events by extending the duration time using 20 times longer DNAs. From this we were able to conclude that the third hypothesis was correct.

In this way, when unexpected events happen, it is interesting to imagine what is happening, to make hypotheses, and to test them. It is most satisfying when the hypothesis is correct, and the causes of effects becomes clear. I am pleased that I was able to solve the problems I encountered one by one, and achieve the results of this paper with the support of many others.

 

Conclusion

I am humbled to have been selected by Nature Nanotechnology. We feel that it would not have been possible to accomplish this research without the accumulated knowledge and technology of the three labs. We were able to achieve the results of this research by referring to many prior papers and utilizing a wide range of technologies obtained through collaborative research. We hope that this research will be used as a precedent to produce new and wonderful research results. We are honored to have our research results accepted by Nature Nanotechnology. We will continue to work hard to have our research accepted by this journal again.

Keisuke Shimizu

_, Tokyo University of Agriculture and Technology