Difference between revisions of "Part:BBa K5150003"

Latest revision as of 13:59, 2 October 2024

Bioactive Multipeptide 1 for B.subtilis

Bioactive multipeptide 1 (Optimized for B.subtilis)

This multipeptide was carefully designed to integrate peptides with antidiabetic, antihypertensive, hypocholesterolemic and antithrombotic properties from food sources such as quinoa, soybean, goat milk, cow milk and calpis. It is optimized to be highly expressed in B.subtilis enabling straightforward purification through affinity chromatography.

The main objective is to encapsulate the protein for oral ingestion so it could be released in the gut. Once released, the bioactive peptides are absorbed into the bloodstream, where they can modulate key metabolic pathways linked to diabetes and its complications. The protein can be used in projects related to nutrition studies, therapeutic natural products, food development or investigation of digestive proteases.

Source

The combination of the 5 bioactive peptides that were chosen was based on their similar values half maximal inhibitory concentration IC50. They were extracted from different food sources such as quinoa, soy, goat milk, cow milk and calpis based on their results reported in literature (Table 1). ^{[1] [2] [3] [4] [5]}

AlphaFold (3D structure)

As a next step, AlphaFold was utilized to predict the 3D structure and it was further refined by ModRefiner. The sequence's 3D model prediction, refinement. This sequence shows a relatively compact and well-folded structure, with some beta-sheets. The 94.2% of its amino acids residues were located in most favored regions. These both factors indicate a well-defined fold, which means protein might have good structural stability, making it likely to be functionally efficient (Figure 1, Figure 3) also it showed a good stability at a pH from 4.5 to 8 (Figure 2).

Molecular Docking

They analyzed the interactions between the multipeptide and pancreatic juice proteases such as trypsin, chymotrypsin C, and pancreatic elastase II. As shown in Figure 1, the sequence functional biopeptides were included in the structure through stick representation, highlighting its surface with different colors.

In the first proposal the color code worked as follows: Red for IQAEGGLT, cyan for LPQNIPPL, green for LLQKW, yellow for IAVPTGVA, and orange for KRDS, each with its specified effect.

According to Figure 1, the protein presents a rigid nuclear well defined structure similar to a conventional protein, which allows structural stability. Stability and molecular interaction tests on computer tools to validate the models, are presented below. Molecular docking was conducted to predict the binding interactions between the designed peptides and their target proteins. This step is critical for confirming the biological activity of the peptides, ensuring that they can interact with target molecules in a stable and functional manner.

The cleavage sites of the proteases trypsin, chymotrypsin C, and pancreatic elastase II with the multi peptides were predicted with the EHP tool of the database DFBP (Database of Food-derived bioactive peptides)(Figure 4).

According to the results, the direct interaction of each enzyme with the peptides was determined so that it could be specified in the docking parameters, which are shown in Figure 5. Also, the catalytic sites of each enzyme were taken from InterPro, and adjusted in txt (file modification on block notes) for more effective results. Each protease was combined with the proposal. Due to the above, a total of 3 interactions were predicted with the software Haddock 2.4. From the proposal, Haddock created between five and twelve clusters with four different structures each. The best three clusters were chosen, as well as the best figure of each one in order to have three main representations.

For the first proposal the results of interactions are shown in Figure 6. where the clusters are specified, as well as its number of interactions, represented with a horizontal red bar chart. For this proposal hydrogen bonds (conventional hydrogen bonds, carbon hydrogen bond), electrostatic (Pi anion, pi cation, attractive charge), electrostatic hydrogen bond (Salt bridge/attractive charge, Pi-Cation), and hydrophobic (Alkyl, pi-alkyl, pi-pi T-shaped, amide pi stacked) interactions were observed. All of these types of bonds are involved in protein-protein interactions.

These results are illustrated in Figure 7, where enzymes are represented in blue color for pancreatic elastase II (PEII), in purple for trypsin (TRY), and in pink for chymotrypsin C (CHY). The peptides are represented with different colors, already established in Figure 1 which correspond to the first proposal.

In the first row the interaction between the first proposal multipeptide (FP) and pancreatic elastase II (PEII) is shown in three figures with the letters A, B, and C, each circle corresponds to a zoom where the interaction can be seen clearly, and the specific regions where the protease interacts with the biopeptide can be observed, making cutting possible. The protein-protein interactions in every figure are represented through sticks with surface enhancement. Direct interactions with the cyan (LPQNIPPL), yellow (IAVPTGVA), and orange (KRDS) peptides were observed, as well as interactions in other regions different from the active peptides. In order to know exactly where the interaction took place, the active residues were previously established in table 1. In the second row, the interactions between the first proposal (FP) multipeptide and trypsin (TRY) are represented in the three clusters with the letters D, E, and F, which present interactions with the orange (KRDS), and red (IQAEGGLT) peptides, as well as with other regions. Finally, in the third row the interactions between chymotrypsin (CHY) and the first proposal multipeptide are represented in the clusters with the letters G, H, and I, where interactions between the cyan (LPQNIPPL), orange ((KRDS), and yellow (IAVPTGVA) peptides with the protease were obtained, as well as with other protein parts.

References

1. ↑ McSweeney, P. L., & O'Mahony, J. A. (Eds.). (2015). Advanced dairy chemistry: volume 1B: proteins: applied aspects. springer.
2. ↑ Vilcacundo, R., Martínez-Villaluenga, C., & Hernández-Ledesma, B. (2017). Release of dipeptidyl peptidase IV, α-amylase and α-glucosidase inhibitory peptides from quinoa (Chenopodium quinoa Willd.) during in vitro simulated gastrointestinal digestion. Journal of Functional Foods, 35, 531-539. h
3. ↑ Hernández-Ledesma, B., Miguel, M., Amigo, L., Aleixandre, M. A., & Recio, I. (2007). Effect of simulated gastrointestinal digestion on the antihypertensive properties of synthetic β-lactoglobulin peptide sequences. Journal of Dairy Research, 74(3), 336-339
4. ↑ Valeriy V. Pak, Shomansur Sh. Sagdullaev, Aleksandr V. Pak, Olim K. Khojimatov. (2023).Modeling of hydrophobic tetrapeptides as a competitive inhibitor for HMG-CoA reductase. Journal of Molecular Structure. Volume 1293.
5. ↑ Rutherfurd, K. J., & Gill, H. S. (2000). Peptides affecting coagulation. British Journal of Nutrition, 84(S1), 99–102. doi:10.1017/S0007114500002312

Sequence and Features