Parts added to Registry of Standard Biological Parts

Multipeptide graphic representation

The previously obtained structures for each proposal were modeled in Alpha fold, and the graphic representation for each sequence was done in PyMol in order to visualize the protein and the assembly of peptides. As mentioned in other sections, three proposals were evaluated, and developed in the following analysis. As shown in figure 1, for every proposal the sequence functional biopeptides were included in the structure through stick representation, highlighting its surface with different colors; for 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.

On the other hand, for the second proposal sequence, which is represented in figure 2, the active peptides are highlighted with colors as follows: Red for LLF, orange for LQKW, yellow for VPP, green for VPP, and cyan for LPQNIPPL, each with its corresponding effect.

Finally, for the third proposal sequence, shown in figure 3, the active peptides are highlighted as follows: Red for DKDYPK, cyan for LPQNIPPL, orange for KRDS, yellow for IAVPTGVA, and green for LLF, each one with its corresponding effect.

According to the three illustrations the first and third proposals presented a rigid nuclear well defined structure similar to a conventional protein, which allows structural stability. On the other hand, the second proposal is unfolding, which may not be favorable for a good stability. However, it is necessary to perform stability and molecular interaction tests on computer tools to validate the models, these tests are presented below.

Docking results

Molecular docking is a key tool in structural molecular biology. The goal of ligand-protein docking is to predict the predominant binding modes of a ligand with a protein of known three-dimensional structure. Successful docking methods search high dimensional spaces effectively and use a scoring function that correctly ranks candidate dockings (Morris & Lim-Wilny, 2008). 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 [32].

Results: For each proposal, 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), which is already reported in the engineering section “9. Test: Protease Cleavage Site Prediction”. 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 table 1. 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 every proposal, so the active residues were different for each sequence according to their interaction with the enzyme. Due to the above, a total of nine interactions were predicted with the software Haddock 2.4.

From each 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 table 2. 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 4, 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.

For the second proposal the results of interactions are shown in table 3. where the clusters are specified, as well as its number of interactions, represented with a horizontal green 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-Donor Hydrogen bond), Sulfur-X, and hydrophobic (Alkyl, pi-alkyl, pi-pi T-shaped) interactions were observed to be involved in protein-protein interactions.

These results are illustrated in figure 5. 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 figures 1, 2, and 3, which correspond to the second proposal. In the first row the interaction between the second proposal (SP) multipeptide 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, just as in figure 4. Direct interactions with the red peptide (LLF) were observed, as well as interactions in other regions different from this peptide which are grey colored. In the second row, the interactions between the second proposal (SP) multipeptide and trypsin (TRY) are represented in the three clusters with the letters D, E, and F, also presenting an interaction with the yellow (VPP), and orange (LQKW) peptides, as well as with other protein regions represented in grey. Finally, in the third row the interactions between chymotrypsin (CHY) and the second proposal (SP) multipeptide are represented in the three clusters with the letters G, H, and I, where direct interactions of the protease with the green (VPP), yellow (VPP), and orange (LQKW) peptides were observed.

For the third proposal the results of interactions are shown in table 4, where the clusters are specified, as well as its number of interactions, represented with a horizontal yellow bar chart. 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 6. 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 figures 3, which corresponds to the third proposal. In the first row the interaction between the third proposal (TP) multipeptide 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, just as in figures 4, and 5. 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 third proposal (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 between chymotrypsin (CHY) and the third proposal (TP) multipeptide 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.

The subsequent experiments are described in the Wetlab section.