Biopanning

The indirect ELISA process that screens for a nanobody that binds best with the target bacteria, resulted in a clone that did not have any genes. A potential reason could be that the nanobody gene sent by Chulalongkorn University only had a vector, thus no protein coding sequence could be identified. Though, if more clones than just the twenty sent were screened, a nanobody with a gene and sequence would have been identified. Furthermore, the prepared phage library was old, therefore, the chances of identifying a nanobody gene was even lower.

Modified Bombolitin Modeling

The bombolitin antimicrobial peptide is already established to be an antimicrobial peptide that has the ability to penetrate cell membranes and cause cell lysis. Our project aims to modify the structure of the bombolitin through the addition of three amino acids in order to increase the peptide's hydrophobicity and ability to penetrate the Ralstonia Solanacearum cell membranes. There are various possibilities in which the three additional amino acids can be ordered. Although it is possible to express and perform in-vitro testing for all the combinations, it is much easier to check the most likely combinations through modeling and molecular docking.

Molecular Model and Docking

We utilized HADDOCK and Alphafold programs to model the peptide and perform molecular docking. After modeling the peptide structure, we end up with the target, bombolitin and protein target, and cell membrane. From this, we are able to find the specific bind, the type of cell protein, and binding affinity of our modeled peptide modification.

Results

Bombolitin with LLL Modification Structure Prediction

Analysis

As a result of altering the structure of the bombolitin through adding additional amino acids, the modeling and calculations from molecular docking shows that the modified version LLL is the structure with the highest efficiency. The enthalpy levels of the LLL modification is the lowest when compared to other versions. The more negative the enthalpy, the higher the affinity the peptide has in regards to being efficient in penetrating the membrane.

Future Work

By adding three amino acids and changing the structure in the program, the efficiency of the modified bombolitin is determined by the given polarity, hydrophobicity, and hydrophilicity. Visualizing the testing of the efficiency of the novel design through modeling before experimentation was very beneficial when taking the time constraints into consideration. However, lab experimentation is very essential to test the applications of the peptide. To conclude, the binding energy and the model have been utilized throughout the lab as reference.

Reference

[1] Jumper, J et al. Highly accurate protein structure prediction with AlphaFold. Nature (2021).