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Modelling

See the theoretical framework created to predict, design, and validate our project.

Full Length PbEL04

  • FL-PbEL04 is highly expressed during gall formation in Clubroot
  • Its molecular functions remain largely uncharacterized
  • Contains 3 regions for ideal protein-protein binding interactions based on the electrostatic potential Epitope

Legend:

  • Purple (Epitope A)
  • Pink (Epitope B)
  • Green (Epitope C)

Video 1.0: Video of full-length PbEL04 three-dimensional (3D) molecular simulation and colouring of the 3 target epitope regions for the chimeric protein fluorescent probes

  • Docking Simulation of Full Length PbEL04 and Chimeric Anti-PbEL04 GFP Protein

    Legend:

    • Dark Blue - FL-PbEL04 Epitope Sequence A
    • Pink -FL- PbEL04 Epitope Sequence B
    • Blue- FLP-PbEL04 epitope sequence C
    • Green- CAPE-GFP
    • Yellow- CAPE-GFP Paratope 1
    • Orange- CAPE-GFP Paratope 2

    Supplementary Video 1.0: Video of docking simulation of the top 5 predictions of FL-PbEL04 and Chimeric Anti-PbEL04 GFP Protein (CAPE-GFP), it is denoted as C2 in the video this was the dry lab code for this protein, based on outputted GRAMM results. The third prediction was the best prediction due to contacts made, scoring and location on the receptor.

Electrostatic Interactions of Full-Length PbEL04 Binding with Chimeric Anti-PbEL04 GFP Protein (Paratope 1)

Table 1.0

Table 1.0: Table shows contacts displayed between the Electrostatic Interaction of Epitope B (Pink) residue in FL-PbEL04 and Paratope 1 (Yellow) residues in CAPE-GFP, as indicated in Video 2.0 (below). The classification of each amino acid is shown in brackets. The total number of contacts achieved was 12 calculated using UCSF Chimera.

Figure 1.0: Image of Full-length PbEL04 (FL-PbEL04) and Chimeric Anti-PbEL04 GFP Protein (CAPE-GFP) residues interacting; Video 2.0: (top left)Video of FL-PbEL04 Epitope A (Pink) and CAPE-GFP Paratope 1 (Yellow) showing electrostatic interactions, as displayed in Table 1.0; Video 2.1: (top right) Video of FL-PbEL04 epitope B ((Pink) and CAPE-GFP Paratope 1 (Yellow) showing electrostatic interactions in a cloud-like form. Blue (+) and Red (-) regions show the polarity of interacting side chains.

Figure 1.1

Figure 1.1: Graph showing interactions between Full Length PbEL04 (Pink residues) and Chimeric Anti-PbEL04 GFP Protein (CAPE-GFP, Yellow residues) Paratope 1, as displayed in Table 1.0.

Hydrogen Bond Results for CAPE-GFP Paratope 1

Table 2.0

Table 2.0: Table shows hydrogen bonds displayed between the Electrostatic Interactions of Epitope B (Pink) residues in FL-PbEL04 and Paratope 1 (Yellow) residues in Chimeric Anti-PbEL04 GFP Protein (CAPE-GFP, Green), as showcased in Video 3.0 (below). These do not include intra-molecular bonding. The classification of each amino acid is displayed in brackets. The total number of Hydrogen bonds achieved was 3.

Figure 2.0: Image of FL-PbEL04 Epitope B (Pink) showing hydrogen bonds between CAPE-GFP Paratope 1 (Yellow) and CAPE-GFP residues (Green), there are 3 hydrogen bonds formed; Video 3.0: Video of Hydrogen Bonds between FL-PbEL04 and CAPE-GFP show 3 total hydrogen bonds, as shown in Table 2.0.

Electrostatic Interactions of FL- PbEl04 and CAPE-GFP Paratope 2

Table 3.0

Table 3.0: Table shows contacts displayed between the Electrostatic Interaction of Epitope B (Pink) residues in FL-PbEL04 and Paratope 2 (Orange) residues in CAPE-GFP, as shown in Video 4.0. The classification of each amino acid is displayed in brackets. The total number of contacts achieved was 39 calculated using UCSF Chimera. Vasker Labs predicted a binding score of 480.

Figure 3: Image of Full-length PbEL04 (FL-PbEL04) and Chimeric Anti-PbEL04 GFP Protein (CAPE-GFP) residues interacting; Video 4.0: FL-PbEL04 Epitope A (Pink) and CAPE-GFP Paratope 2 (Orange) showing electrostatic interactions, as displayed in Table 3.0; Video 4.1: FL-PbEL04 Epitope A (Pink) and CAPE-GFP Paratope 2 (Orange) showing electrostatic interactions in a cloud-like form. Blue (+) and Red (-) regions show the polarity of interacting side chains.

Figure 3.1

Figure 3.1: Graph showing interactions between Full Length PbEL04 and Chiemric Anti-PbEL04 GFP Protein (CAPE-GFP) Paratope 2, as displayed in Table 3.0.

Note: No intramolecular hydrogen bonds were found between Epitope B (FL-PbEL04) and Paratope 2 (CAPE-GFP)

  • Docking simulation of Chimeric Anti-Pbel04 Fluorescent Probe (CAPE-AFP)

    Legend:

    • Purple- PbEL04 Epitope Sequence A
    • Pink - PbEL04 Epitope Sequence B
    • Light Blue - PbEL04 Epitope Sequence C
    • Orange - CAPE-AFP
    • Yellow- CAPE-AFP Paratope 1
    • Red - CAPE-AFP Paratope 2
    • Green- CAPE-AFP Paratope 3

    Supplementary Video 2.0: Docking Simulation of the top 5 predictions of FL-PbEL04 docking simulation with Chimeric Anti-PbEL04 Florophore Probe (CAPE-AFP), based on GRAMM results. The fourth orientation was the best prediction due to the contacts made, scoring and location on the receptor.

    Note: No contacts were made between PbEL04 and Paraope 1 of CAPE-AFP

Electrostatic Interactions of FL-PbEL04 and CAPE-AFP (Paratope 2)

Table 4.0

Table 4.0: Table shows contacts displayed between the Electrostatic Interaction of Epitope B (Pink) residues in FL-PbEL04 and residues in CAPE-AFP Paratope 2 (Red). There were a total of 90 contacts predicted using UCSF chimera, as indicated in Video 5.0 (below). The classification of each amino acid is displayed in brackets. Vasker Labs GRAMM application predicted a score of binding at 399.

Figure 4.0: Image of FL-PbEL-04 Epitope B (Pink) and CAPE-AFP Paratope 2 (Red) showing electrostatic interactions; Video 5.0: Video of FL-PbEL04 Epitope B (Pink) and CAPE-AFP Paratope 2 (Red) showing electrostatic interactions, as displayed in Table 4.0.

Figure 4.1

Figure 4.1: Graph showing interactions between Full Length PbEL04 Epitope B (Pink residues) and Chimeric Anti-PbEL04 Fluorescent Probe (CAPE-AFP, Red residues) Paratope 2, as displayed in Table 4.0.

Hydrogen Bond Results for CAPE-AFP Paratope 2

Table 5.0

Table 5.0: Table shows Hydrogen Bonds displayed between the Electrostatic Interactions of Epitope B (Pink) residues in FL-PbEL04 and Paratope 2 (Red) residues in Chimeric Anti-PbEL04 Fluorescent Probe (CAPE-AFP), as showcased in Video 6.0. These do not include intra-molecular bonding. The classification of each amino acid is displayed in brackets. The total number of Hydrogen bonds achieved was 3.

Figure 5.0: Image of FL-PbEL04 Epitope B (Pink) showing hydrogen bonds between CAPE-AFP Paratope 2 (Red), there are 3 hydrogen bonds formed; Video 6.0: Video of Hydrogen Bonds between FL-PbEL04 and CAPE-AFP show 3 total hydrogen bonds, as shown in Table 5.0.

Electrostatic Interactions of FL-PbEL04 and CAPE-AFP (Paratope 3)

Table 6.0

Table 6.0: Table shows contacts displayed between the Electrostatic Interaction of Epitope B (Pink) residues in FL-PbEL04) and residues in CAPE-AFP Paratope 3 (Green). There were a total of 32 contacts predicted using UCSF chimera, as indicated in Video 7.0 (below). The classification of each amino acid is displayed in brackets. Vasker Labs GRAMM application predicted a score of binding at 399.

Figure 6.0: Image of FL-PbEL-04 Epitope B (Pink) and CAPE-AFP Paratope 3 (Green) showing electrostatic interactions; Video 7.0: Video of FL-PbEL04 Epitope B (Pink) and CAPE-AFP Paratope 3 (Green) showing electrostatic interactions, as displayed in Table 6.0.

Figure 6.1

Figure 6.1: Graph showing interactions between Full Length PbEL04 Epitope B (Pink residues) and Chimeric Anti-PbEL04 Fluorescent Probe (CAPE-AFP, Green residues) Paratope 3, as displayed in Table 6.0.

Hydrogen Bond Results for CAPE-AFP Paratope 3

Table 7.0

Table 7.0: Table shows Hydrogen Bonds displayed between the Electrostatic Interactions of Epitope B (Pink) residues in FL-PbEL04 and Paratope 3 (Green) residues in Chimeric Anti-PbEL04 Fluorescent Probe (CAPE-AFP), as showcased in Video 8.0 (below). These do not include intra-molecular bonding. The classification of each amino acid is displayed in brackets. The total number of Hydrogen bonds achieved was 1.

Figure 7.0: Image of FL-PbEL04 Epitope B (Pink) showing hydrogen bonds between CAPE-AFP Paratope 3 (Green), there is 1 hydrogen bond formed; Video 8.0: Video of Hydrogen Bonds between FL-PbEL04 and CAPE-AFP show 1 hydrogen bond, as shown in Table 7.0.

Electrostatic Interactions of CAPE-AFP

Figure 7.0: Image of FL-PbEL04 Epitope B (Pink) showing hydrogen bonds between CAPE-AFP Paratope 3 (Green), there is 1 hydrogen bond formed; Video 9.0: Video of FL-PbEL04 Epitope B (Pink) and CAPE-AFP Paratope 2 (Red) showing electrostatic interactions in a cloud-like form. Blue (+) and Red (-) regions show the polarity of interacting side chains.

TEM Validation of PbEL04’s Structure

TEM validation of PbEL04 structure

Figure 8.0: On the left, the computationally determined model from AlphaFold showcases the predicted fibrillar structure. On the right, the transmission electron micrograph of PbEL04 with a thyrodexin tag validates this fibrillar morphology. The varying lengths and occasional bundling of the fibril monomers can also be observed against the negatively stained background.

Discussion

After comparing the data acquired from the docking simulations and the contact information, the dry lab determined which prediction provided the best binding results for our chimeric fluorescent probes. For the Chimeric Anti-PbEL04 GFP Protein (CAPE-GFP) the best prediction was determined to be the third orientation outputted from Vasker Labs GRAMM docking application. This orientation had our chimeric protein bound with Full Length PbEL04 at Epitope B, as showcased in Supplementary Video 1.0, and provided a score of 470. This score indicated that our proteins are highly likely to bind in this manner. Through using UCSF Chimera, a total of 51 contacts were found. There were 12 contacts in Paratope 1, as described in Table 1.0, and in Paratope 2 it yielded 39 contacts, as described in Table 3.0. Videos 2.0 and Videos 4.0 highlight these exact residues interacting between FL-PbEL04 and CAPE-GFP. Along with this, UCSF chimera provided the Hydrogen Bonds observed between each Paratope. For CAPE-GFP Paratope 1 with Epitope B in FL-PbEL04 there were 3 observed H bonds, as observed in Table 2.0 and Video 3.0 In Paratope 2, 0 hydrogen bonds were observed. Finally, the Electrostatic Interactions are shown in Video 2.1 and Video 4.1, they help demonstrate these contacts more efficiently. The cloud-like videos help show the varying polarity interactions from the side chain residues. Overall, our results provide high confidence that the chimeric protein and receptor will bind inimitably. The number of contacts observed between residues as well as the visual interactions of the receptor linking with the chimeric protein are promising. This protein complex was the proof of concept to understand if the system would work. From here, tests were begun on the Chimeric Anti-PbEL04 Fluorophore Probe, CAPE-AFP. Docking simulations were once again run in Vasker Labs GRAMM docking application and it was compared to the binding information gained through UCSF chimera. The top prediction outputted from GRAMM was the fourth prediction, which had a score of 399 and is showcased in Supplementary Video 2.0. This score, while lower than CAPE-GFP, still indicates a high likelihood of binding and the difference in scores may be due to the variance of electrostatic interactions that may be occurring between proteins. Through UCSF chimera, a total of 122 contacts were found between Paratope 2 and Paratope 3 of CAPE -FP and Epitope B of full-length PbEL04. In Paratope 2, 90 contacts were found, in Table 34.0 and Video 5.0, along with 3 hydrogen bonds, as shown in Table 5.0 and Video 6.0. In Paratope 3, 32 contacts were found, Table 6.0 and Video 7.0, and 1 hydrogen bond, as displayed in Table 7.0 and Video 8.0. Finally, the electrostatic interactions between FL-PbEL04 and CAPE-AFP Paratope 2 and Paratope 3 can be visualized in a 3D model, as shown in Video 9.0. The cloud-like electrostatic interactions show areas of high polarity between the proteins in the Blue (+) and Red (-) regions. Overall, our results for CAPE-AFP are very favourable and show a high probability of binding in a lab. Furthermore, it’s worth noting while CAPE-GFP had a higher GRAMM docking score, the CAPE-AFP demonstrated a significantly greater number of contacts with its respective epitope. This suggests that while GRAMM docking scores provide a general gauge of binding potential, the true complexity of protein-protein interactions, including the intricacies of residue contacts and electrostatics, require a more nuanced analysis. As we proceed, we will continue to validate these in-silico results with in-vitro assays to corroborate our findings.

Future Directions

  • Use molecular dynamic simulations to predict its behaviour in varied environmental circumstances

Dry Lab Methods

Generate Complementary Amino Acid Sequence with Chimeric binding Regions

The Wet Lab engineered a compementary Amino Acid Sequence to PbEL04 and added on specific chimeric binding regions

3D Models and PDB

Input engineered amino acid sequence of PbEL04 into Aplha Fold Outputs 3D model and PDB to use for later steps

Docking Simulations

Input PDB files into Vasker Labs GRAMM application Set parameters t0 100,000 oreientations outputting top 5 docking results

Binding analysis

Input Docking simulation predictions into UCSF Chimera Create movies for binding and get electrostatic interactions and hydrogen bond contact data

Dry Lab Software

Alphafold, GRAMM, and UCSF Chimera are pivotal tools in the field of molecular biology, with Alphafold specializing in protein structure prediction, GRAMM in molecular docking simulations, and UCSF Chimera in visualization and interaction analysis.

Alphafold

Background: Developed by DeepMind, Alphafold is a state-of-the-art tool in the field of protein structure prediction. It has revolutionized the understanding of proteins, their folding, and functionality.

How it Works: Utilizes deep learning and extracts knowledge from publicly available protein data repositories.

Confidence Measurement (pLDDT): The tool offers an inherent confidence score for each prediction it makes on a scale of 0-100.

  • Very High Confidence (Dark Blue): Score > 90, indicating a highly reliable protein structure prediction.

  • Confident (Light Blue): Score between 70 and 90, suggesting a reasonably accurate structure prediction.

  • Low Confidence (Yellow): Score between 50 and 70, indicating a need for caution or further validation.

Figure 9.0

Figure 9.0: PbEL04’s structure with AlphaFold’s measure of model confidence, Dark Blue is areas of high confidence for binding, Light Blue is confident, Yellow is low confidence and Orange is no confidence of binding.

GRAMM (by Vasker Labs)

Background: GRAMM is a molecular docking software. Docking simulations allow researchers to predict how two molecules, such as a drug and its target protein, might fit together.

Features:

  • A 6-dimensional search: This means the tool is considering three translational and three rotational parameters to find the best fit between the molecules.

  • Orientation Considerations: GRAMM evaluates 100,000 potential orientations to ensure a comprehensive search.

  • Top 5 Results: To provide users with multiple potential fits, the tool outputs the five most promising orientations.

  • Scoring System: This assists researchers in assessing how likely two molecules are to bind.

    • Unlikely: Score less than 200 suggests a poor fit.

    • Likely: Scores between 300-400 hint at a favorable interaction.

    • Highly Likely: Score greater than 400 indicates a very promising binding interaction.

    Figure 10.0

    Figure 10.0: Full Length PbEL04 and Chimeric Anti-PbEL04 GFP Protein top 5 docking predictions from Vasker Labs GRAMM

UCSF Chimera

Background: UCSF Chimera is a highly regarded visualization software developed by the University of California, San Francisco. It’s frequently used in molecular biology and bioinformatics.

Functionality: Allows for:

  • Visualization: Users can view 3D structures of molecules, aiding in understanding and presentation.

  • Electrostatic Interaction Analysis: Chimera can predict and showcase areas of positive, negative, and neutral charges, which is essential for understanding molecular interactions.

  • Movies: Users can create animated representations, helpful for presentations or understanding dynamic processes.

image of PbEL04’s structure with AlphaFold’s measure of model confidence

Figure 11.0. PbEL04’s structure with AlphaFold’s measure of model confidence, Dark Blue represents areas of high confidence for binding, Light Blue is confidence, Yellow is low confidence, and Orange is no confidence of binding.

Supplementary Information

  • Supplementary Figure 1.0

    Supplementary Figure 1.0: Dry lab classification codes for Master Movies, the connection to the present naming system is displayed in Supplementary Table 1. Un-optimized proteins were not used after these initial stages, the protein system was upgraded to optimized which involved the rearrangement of tags and other sequences,

  • Supplementary Table 1.0

    Supplementary Table 1.0: Master table for the protein information used by the dry lab. Under the column labelled “Dry Lab Names”, the number 1 denotes the Unoptimized version of the protein and the number 2 denotes the Optimized version of the protein. This table also shows the binding specificity of the proteins. Bi-specific implies that it can bind with both Full Length PRO-1 and Full Length PbEL04. There are two different hex codes, the one specified as “Chimera”, is the hex code uniquely used in the UCSF Chimera application, the regular 6-digit hex codes are universal.

  • Supplementary Video 3.0: Video of Docking simulation of the top 5 predictions of Full Length PRO1 (FL-PRO1) and Chimeric Anti-PRO1 Fluorescent Protein (CAP-FP), based on outputted GRAMM results, it is denoted as GFP-D2 (optimized) in the video this was the dry lab code for this protein.

    Supplementary Video 4.0: Video of Docking simulation of the top 5 predictions of Full Length PbEL04 (FL-PbEL04) and Chimeric Anti-Bispecific GFP Protein (CAB-GFP), based on outputted GRAMM results, it is denoted as E2 (optimized) in the video this was the dry lab code for this protein.

    Supplementary Video 5.0: Video of Docking Simulation of the top 5 predictions of FL-PbEL04 docking simulation with Recombinant Chimeric Probe with Mouse Antibody Framework (Rec. CP-MAF), based on GRAMM results. It is denoted as F2 (optimized) in the video this was the dry lab code for it.

    Supplementary Video 6.0: Video of Docking simulation of the top 5 predictions of Full Length PRO1 (FL-PRO1) and Chimeric Anti-PRO1 Fluorescent Probe (CAP-FLP), based on outputted GRAMM results, it is denoted as FLU-G2 (optimized) in the video this was the dry lab code for this protein.

  • Supplementary Video 7.1: Master Movie of Full Length PbEL04, Dry lab code was A1 (Un-Optimized), refer to Supplementary Table 1.0.

    Supplementary Video 8.1: Master Movie of Full Length PRO1, Dry lab code was B1 (Un-Optimized), refer to Supplementary Table 1.0.

    Supplementary Video 9.1: Master Movie of GFP, dry lab code C1 (Un-Optimized) protein that binds specifically to FL-PbEL04. This version is the GFP protein without the added His Tags, Its counterpart is shown in Supplementary Video 9.2, refer to Supplementary Table 1.0.

    Supplementary Video 10.1: Master Movie of GFP, dry lab code D1 (Un-Optimized) protein that binds specifically to PRO1. Refer to Supplementary Table 1.0

    Supplementary Video 11.1: Master Movie of GFP, dry lab code E1 (Un-Optimized) protein that is bi-specific for FL-PbEL04 and FL-PRO1, refer to Supplementary Table 1.0

    Supplementary Video 12.1: Master Movie of Recombinant Chimeric Probe with Mouse Antibody Framework, dry lab code F1 (unoptimized). It is bi-specific for FL-PbEL04 and FL-PRO1. Refer to Supplementary Table 1.0

    Supplementary Video 13.1: Master Movie of a Fluorophore protein, dry lab code G1 (Un-Optimized) that binds to PRO1, refer to Supplementary Table 1.0

    Supplementary Video 14.1: Master movie of a Fluorophore that binds specifically to FL-PbEL04. This version does not have the linkers or the added-on histidines, dry lab code of K1. The docking simulation is shown in Supplementary Video 2.0.

  • Supplementary Video 9.2: Master Movie of the Chimeric Anti-PbEL04 GFP Protein (CAPE-GFP), Dry lab code C2. This protein is shown to bind in Supplementary Video 1.0, refer to Supplementary Table 1.0.

    Supplementary Video 10.2: Master Movie of Chimeric Anti-PRO1 Fluorescent Protein (CAP-FP), dry lab code D2. This protein is shown to bind in Supplementary Video 3.0, refer to Supplementary Table 1.0.

    Supplementary Video 11.2: Master Movie of Chimeric Anti-bispecific GFP (CAB-GFP) protein, dry lab code E2 (Optimized). This protein is shown to bind in Supplementary Video 4.0, refer to Supplementary Table 1.0

    Supplementary Video 12.2: Master Movie of Recombinant Chimeric Probe with Mouse Antibody Framework (Rec. CP-MAF), dry lab code F2 (Optimized). This protein is shown to bind in Supplementary Video 5.0, refer to Supplementary Table 1.0

    Supplementary Video 13.2: Master Movie of Chimeric Anti-PRO1 Fluor Protein (CAB-FLP), dry lab code G2 (Optimized). This protein is shown to bind in Supplementary Video 5.0, refer to Supplementary Table 1.0

  • EpitopeSequenceParatopeSequence
    ATGNGVVCTDVDECRANNGG1VIIRILSCERLRKCDSQQIIC
    BFAGDGLTCKDVDECRTNNGGC2VTIQILDCRRIQKCLLQQII
    CFSGNGIRCDDVNECLLNNGG-

    Supplementary Table 2.0: Epitope and Paratope Amino Acid Sequences for Full Length PbEL-04

  • EpitopeSequenceParatopeSequence
    ADDENKVKQAYNGDLTDLQKREFEK1VLDSMDMQEDLEDIGK
    BPGVNTAGGSGSDVGT2KKRQDVDNIYQIKISKLNDERVRD
    CGLRTMRMNKRPKHLAE-

    Supplementary Table 3.0: Epitope and Paratope Amino Acid Sequences for Full Length PRO-1