Part:BBa_K5150000
Bioactive multipeptide 3 (Optimized for E.coli)
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 E.coli 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 (Figure 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 (Figure 2). It has some structured regions, such as several beta-sheets and an alpha-helix but there are also extended unstructured regions, so this mixture suggests moderate stability. The 91.3% 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 2, Figure 4) also it showed a good stability at a pH from 4.0 to 8 (Figure 3).
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 5).
Molecular Docking
There were analyzed the interactions between the multipeptide and pancreatic juice proteases such as trypsin, chymotrypsin C, and pancreatic elastase II. As shown in Figure 2, the sequence functional biopeptides were included in the structure through stick representation, highlighting its surface with different colors.
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 yellow bar chart (Figure 7). For this proposal hydrogen bonds (conventional hydrogen bonds, carbon hydrogen bond, Pi-Donor hydrogen bond), electrostatic (Pi anion, attractive charge), electrostatic hydrogen bond (Salt bridge/attractive charge), and hydrophobic (Alkyl, pi-alkyl, pi-sigma, amide-pi stacked) interactions were recognized to be involved in the protein-protein interactions.
These results are illustrated in Figure 8. where enzymes are represented in blue color for pancreatic elastase II, in purple for trypsin, and in pink for chymotrypsin C. The peptides are represented with different colors, already established in Figure 2. In the first row the interaction between the multipeptide (TP) and pancreatic elastase II (PEII) is shown in three figures with the letters A, B, and C (Figure 8). Each circle corresponds to a zoom where the interaction can be seen clearly. Direct interactions with the cyan (LPQNIPPL) and green (LLF) peptides were observed, as well as interactions in other regions different from the active peptides. In the second row, the interactions between the multipeptide (TP) multipeptide and trypsin (TRY) are represented in the three clusters with the letters D, E, and F, presenting interactions with the red (DKDYPK), and orange (KRDS) peptides, as well as with other protein regions colored in grey. Finally, in the third row the interactions with chymotrypsin (CHY), are represented in the three clusters with the letters G, H, and I, where direct interactions between the cyan (LPQNIPPL), and red (DKDYPK) peptides were observed.
The docking results indicate that there is an interaction between the digestive enzymes and the protein, this suggests that the protein has recognized one of its cleavage sites, this showed that the multipeptide sequences are a potential substrate for intestinal proteases. Consequently, this confirms that peptide fragments and bioactive peptides will be released during digestion. This interaction is essential for validating that the enzyme-protein binding will result in the hydrolysis of the protein, leading to the liberation of the peptides of interest. However the verification of their release and their functionality require experimental tests.
References
- ↑ McSweeney, P. L., & O'Mahony, J. A. (Eds.). (2015). Advanced dairy chemistry: volume 1B: proteins: applied aspects. springer.
- ↑ Vilcacundo, R., MartÃnez-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
- ↑ 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
- ↑ 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.
- ↑ 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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