Model

1.Our Softwares

Chimera & ChimeraX

These are versatile visualization and analysis tools that allow for the exploration and manipulation of molecular structures. We used them for contact analysis and to observe electrostatic interactions between our models. They offer a wide range of features, including the ability to visualize complex molecular interactions in 3D.

Pros: UCSF Chimera excels in its easy-to-use interface, using straightforward drop-down menus to access tools and features. It also allows for intuitive and simple controls of the camera and manipulation of the protein. Chimera also allows the use of add-ons and custom inputs through Python. Additionally, there is access to built-in databases to ease molecule retrieval. ChimeraX is actively developing with regular updates that introduce new features, methods, and compatibility with research tools. It can handle larger datasets with higher speeds and efficiency than UCSF Chimera.

Cons: UCSF Chimera is an outdated software that no longer receives support from its developers. Due to its older framework, it is less efficient when running more complex systems. Due to its lack of support, there is a higher likelihood of experiencing bugs and glitches. Compared to its successor, ChimeraX, it also has limited visualization quality. ChimeraX has a steeper learning curve and is less intuitive compared to its predecessor. Its features ARE MOSTLY THE SAME but do not allow for backward compatibility with UCSF Chimera. Due to its ongoing development, certain features available in UCSF Chimera are not available in ChimeraX. Because of its recent developments, there is a lack of documentation and tutorials available online for the software.

Vakser Lab GRAMM Docking Simulations

This computational tool is used for predicting protein-protein and protein-ligand interactions, helping us understand how different molecules interact at a structural level. It searches for potential binding orientations and conformations of proteins. The software is available as a web server.

Pros: GRAMM uses a grid-based approach to examine the proteins, producing a relatively fast output compared to other molecular docking programs. GRAMM excels in protein-protein interactions and conformational changes during binding. It has excellent accessibility due to its availability as a website. Its user interface is straightforward and makes it easy to run docking simulations without significant technical knowledge. GRAMM also allows for larger files to use

Cons: GRAMM has a limited amount of options regarding its docking process, which focuses on rougher docking predictions rather than providing high-resolution results – this can be circumvented with the use of visualization software.

GROMACS

This molecular dynamics simulation software is well-suited for simulating the behavior of biological macromolecules. It is known for its efficiency and scalability, GROMACS allows researchers to simulate large systems with high accuracy.

Pros: GROMACS is highly optimized for speed and has a very user-friendly interface that can be used on modern hardware. It is very versatile and can handle a multitude of different molecular systems and environments. Along with this, the program is free to use and there is a wide array of explanatory forums available to aid new users in using the program.

Cons: GROMACS utilizes the hardware capabilities of the computer it is being run on unless there is access to a supercomputer. This can be troublesome since the GPU and processors of the computer may not be strong enough to run the desired simulation. Along with this, it provides limitations on the size of the simulation that can be run which may induce problems observed in the simulation.

AMBER

AMBER is a collection of molecular simulation programs for performing molecular dynamics simulations. It excels with biomolecules and offers advanced force fields to represent molecular interaction, making it a popular choice for studying protein and nucleic acid dynamics.

Pros: AMBER has an extensive set of force fields for biomolecular systems, making it a great tool for DNA and protein-based simulations. It can integrate with various other tools allowing a robust analyzation of data, providing in-depth results.

Cons: There is a free version of AMBER that provides partial usage but to receive the full license it needs to be paid for which can be troublesome for individuals only using the software occasionally. Generally, AMBER is not as fast as GROMACS since it utilizes more memory and integration with other tools. AMBER is also more complex and harder to learn for new users.

AlphaFold2

AlphaFold2 is a Google-based, advanced AI-driven, machine learning tool made to predict protein structures from amino acid sequences. It utilizes the Protein Data Bank (PDB) to search for similar sequence alignments and structural homology of other proteins in the database. The software will then perform Molecular Dynamic simulations and provide a folded protein in its lowest energy confrontation state.

Pros: Uses AI-driven technology allowing increased efficiency, efficacy, and accuracy of the outputted protein structure. It is fast and effective and provides a generalized structure to base research on if access to Cryo-Electron Microscopy or X-ray crystallography is limited. Since it uses a vast database to compare against most structures predicted are highly accurate making this an effective and accessible tool for researchers.

Cons: Since this compares against a database, if there are regions of a protein that are unlike others in the databank there will be inaccurate folding observed. This is a problem for newly discovered or more novel proteins as there is less information readily available for alpha fold to compare against. Along with this, the software does not account for biological context such as native conditions or post-translational modifications that may occur. The tool provides a good baseline but knowledge of structural biology is needed to interpret the results making it more difficult for new users.

Pymol

Pymol is a user-sponsored molecular visualization software used for visualizing and analyzing molecular structures. Pymol is a popular choice among biologists and bioinformaticians since it is a very flexible program with extensive features such as providing support for integration of other software tools.

Pros: The visualization achieved is high-quality and easy to maneuver. There is an extensive community online with tutorials and help pages to make learning the software easier for new users. The software can integrate with various other software and databases making it easy to transfer data.

Cons: There is a steep learning curve when it comes to this software, especially with its extensive features and scripting ability. Large structures or complex visuals may be hard to work with as it will use the resources of your computer making the program lag or quit. While there is good compatibility with other software, the symbol on its own lacks some analysis tools.

2.Our Proteins

pBEL04

PbEL04 is a cysteine-rich, EGF-like secretory protein, with 3 main surface exposed epitope regions as shown below. It is expressed during the initial infection of the disease and is highly expressed during gall formation on the roots of Canola as a result of pathogen infection. This protein appears to be the earliest form of detection for the disease and thus far is unique to the P. Brassicae pathogen.

Video 1.1: Movie of PbEL04 protein with its binding sequences colored in purple, yellow, and pink. Displayed visually and recorded through UCSF Chimera.

Nanobody

The nanobody is a single-domain antibody that has similar functions to antibodies while being significantly smaller in size. We engineered the nanobody with a lysine region that binds effectively to the fluorescent probe and to the gold nanoparticle allowing a “positive” result to be visualized.

Video 1.2: Movie of nanobody protein with binding sequence colored in light gray and lysine region colored in red. Displayed visually and recorded through UCSF Chimera.

FAB Antibody

The FAB(Fragment Antigen-Binding) antibody is a monovalent, chimeric fragment with a high affinity for specific antigens. It is composed of 2 chains, a light, and heavy chain, as a result of lab-driven cleavage.

Video 1.3: Movie of FAB Antibody protein with its binding sequence colored in light gray. Displayed visually and recorded through UCSF Chimera

3.Docking Stimulations

Parameters

In our GRAMM Docking simulations, we used free docking, reduced the output matches to the top 5 models, and increased the number of scan matches to 100,000. Free docking was used to make sure there were no limitations on our proteins’ ability to bind.

PbEL04 and Nanobody

Video 2.1: Results of the GRAMM docking simulation for PbEL04 and the nanobody, models 1 through 5. PbEL04 binding sequences are highlighted and the nanobody binding sequence is displayed in gray. Displayed visually and recorded through UCSF Chimera.

In this movie, PbEL04 cycles through the simulated models. Models 1, 3, and 5 are most important as they showcase direct binding with the desired epitope regions on PbEL04. These models illustrate 3 possibilities of how our target proteins may bind to PbEL04 and through this, the strength of those interactions can be assessed by looking at the amino acid residues that bind between the paratope and epitope regions. Supplementary data concerning electrostatic and hydrophobic interactions can be inferred as well.

PbEL04 and FAB Fragment

Video 2.2: Results of GRAMM docking simulations for PbEL04 and the FAB, models 1 through 5. Binding sequences are highlighted and the nanobody binding sequence is displayed in gray. Displayed visually and recorded through UCSF Chimera.

In this movie, PbEL04 cycles through the simulated models. Despite extensive sampling of the potential interactions, none of the simulated models of the FAB fragment and PbEL04 resulted in successful binding interactions at the epitope binding regions. This seems to be due to the relatively low binding affinity between the targeted epitope regions and the other interacting chains in the molecule.

Contacts of PbEL04 and FAB fragments

Our Fab fragment protein has shown limited success in both wet lab and dry lab experiments. In the wet lab, we faced challenges in achieving significant binding affinity or functional activity, limiting its potential as a viable candidate. Meanwhile, our in silico docking simulations provided further insights, revealing that the Fab fragment failed to establish any meaningful contacts with the epitope binding sequences. This lack of interaction suggests a possible structural or conformational issue, which may be contributing to the overall lack of effectiveness observed in our experimental outcomes. For this reason, we stopped working with our FAB due to its limited success in both labs.

Contacts of PbEL04 and Nanobody

Model 1

Video 2.3: Model 1 GRAMM docking simulation result of PbEL04 and Nanobody. The PbEL04 protein binding sequence is displayed in purple, the Nanobody binding sequence is displayed in gray, atoms are colored and highlighted by heteroatoms, and contacts are displayed with green lines. Displayed visually through UCSF Chimera

Video 2.4: Movie demonstrating GRAMM docking simulation model 1 contacts between PbEL04 and nanobody binding regions. 53 total contacts between atoms are displayed with yellow lines. Binding region of PbEL04 and the nanobody are displayed in purple and gray respectively.

Figure 2.1: The contact analysis results for Model 1 of the nanobodies contacts made to PbEL04 between the epitope and paratope regions. The results were generated using the Vakser Labs GRAMM docking web application and were analyzed using UCSF Chimera. There were a total of 52 contacts made.

Figure 2.15: The overall contact analysis results of various amino acid groups that Model 1 of the Nanobody made to PbEL04 between the epitope and paratope regions. The results were generated using Vakser Labs GRAMM docking web application and the contact results were analyzed using UCSF Chimera.

The figures above show that most contacts between the nanobody and PbEL04 are occurring between residues Glutamine 124 and Aspartate 148. Which are Polar uncharged and Polar Negatively Charged Amino Acids. These graphs focus mainly on the residence of the nanobody. It is important to look at the composition of binding to determine what the driving forces are for binding. In this case, we can determine that more electrostatic interactions are occurring between the epitope and paratope regions due to a lack of non-polar contacts being formed, which are the main driving factors of hydrophobicity. This particular model showed 52 contacts between the 2 structures with no hydrogen bonding occurring.

Figure 2.2: Hydrophobic Interaction diagram of Model 1 of the Docking of the nanobody and PbEL04 generated by Chimera X.

The Hydrophobic Interaction diagram above shows the hydrophobic areas in brown, there are small regions of brown coloring on this image showing that there is limited hydrophobic interaction occurring between the epitope and paratope regions.

Figure 2.25: Electrostatic Interaction diagram of Model 1 of the Docking of the nanobody and PbEL04 generated by Chimera X.

The electrostatic Interaction Diagram above showcases the residue interactions that contribute to the fold and formation of these proteins. The red regions represent a negative charge force and blue represents a positive charge force. If red and blue regions are close together a “pulling” effect will occur on the proteins due to the charges attracting one another. In regions where there are similar colors close together a “repelling” effect will occur causing the residue to push away from one another ultimately affecting the fold and conformation of the protein.

Model 3

Video 2.5: Model 3 GRAMM docking simulation result of PbEL04 and Nanobody. The pbEL04 protein binding sequence is displayed in purple, the Nanobody binding sequence is displayed in gray, atoms are colored and highlighted by heteroatoms, and contacts are displayed with green lines. Displayed visually through UCSF Chimera “

Video 2.6 : Movie demonstrating GRAMM docking simulation model 1 contacts between PbEL04 and nanobody binding regions. 5 total contacts between atoms are displayed with yellow lines. Binding regions of PbEL04 and the nanobody are displayed in purple and gray respectively.

Figure 2.3: The contact analysis results for Model 3 of the nanobodies contacts made to PbEL04 between the epitope and paratope regions. The results were generated using the Vakser Labs GRAMM docking web application and were analyzed using UCSF Chimera. A total of 5 contacts were recorded.

Figure 2.35: The overall contact analysis results of various amino acid groups that Model 3 of the Nanobody made to PbEL04 between the epitope and paratope regions. The results were generated using Vakser Labs GRAMM docking web application and the contact results were analyzed using UCSF Chimera.

The figures above show the contact analysis of the paratope and epitope regions observed on a nanobody and PbEL04. The total number of contacts observed was 5 with the most dominant being Glutamine 124 and Aspartate 148 on the paratope and epitope. The majority of residue types observed making contacts from the nanobody to PbEL04 were Polar Uncharged. Once again, this provides an inference that the majority of interactions occurring are electrostatic interactions.

Figure 2.4: Hydrophobic Interaction diagram of Model 3 of the Docking of the nanobody and PbEL04 generated by Chimera X.

Figure 2.45: Electrostatic Interaction diagram of Model 3 of the Docking of the nanobody and PbEL04 generated by Chimera X.

On this diagram it appears there is minimal electrostatic interaction occurring due to the muted coloring; this may be due to there only being 5 observed contacts between the paratope and epitope. There is also 1 hydrogen bond observed.

Model 5

Video 2.7: Model 5 GRAMM docking simulation result of PbEL04 and Nanobody. The PbEL04 protein binding sequence is displayed in purple, the Nanobody binding sequence is displayed in gray, atoms are colored and highlighted by heteroatoms, and contacts are displayed with green lines. Displayed visually through UCSF Chimera “

Video 2.8: Movie demonstrating GRAMM docking simulation model 1 contacts between PbEL04 and nanobody binding regions. 14 total contacts between atoms are displayed with yellow lines. Binding regions of PbEL04 and the nanobody are displayed in purple and gray respectively.

Figure 2.5: The contact analysis results for the Model 5 of the nanobodies contacts made to PbEL04 between the epitope and paratope regions. The results were generated using Vakser Labs GRAMM docking web application and were analyzed using UCSF Chimera. This graph demonstrates the 14 contacts that were observed.

Figure 2.55: The overall contact analysis results of various amino acid groups that Model 5 of the Nanobody made to PbEL04 between the epitope and paratope regions. The results were generated using Vakser Labs GRAMM docking web application and the contact results were analyzed using UCSF Chimera.

The figures above show that most contacts between the nanobody and PbEL04 are occurring between residues Leucine 118 and Arginine 146 as well as Aspartate 122 and Aspartate 149 . Leucine is a non-polar amino acid meaning it is expected to be hydrophobic, arginine is a positively charged amino acid and finally aspartate as previously mentioned is a negatively charged amino acid. This contact analysis shows that there are equal hydrophobic and electrostatic interactions occurring on this model in accordance with the amino acid residues interacting between the epitope and paratope regions. These graphs are focussing mainly on the residues of the nanobody. It is important to look at the composition of binding to determine what the driving forces are. This particular model showed 14 contacts between the 2 structures with 2 hydrogen bonds occurring.

Figure 2.6: Hydrophobic Interaction diagram of Model 5 of the Docking of the nanobody and PbEL04 generated by Chimera X.

Figure 2.65: Electrostatic Interaction diagram of Model 5 of the Docking of the nanobody and PbEL04 generated by Chimera X.

On this diagram it appears there is minimal electrostatic interaction occurring due to the muted coloring; this may be due to there being 14 observed contacts between the paratope and epitope. There are also 2 hydrogen bonds observed.

Comparing Contacts Across All Supporting Models

Figure 2.7: The contact analysis results for the Nanobodies contacts made to PbEL04 between the epitope and paratope regions. Model 1 (blue), model 3 (orange) and model 5 (green) were generated using Vakser Labs GRAMM docking web application and the contact results were analyzed using UCSF Chimera.

Figure 2.75: The overall contact analysis results of various amino acid groups that the Nanobodies contacts made to PbEL04 between the epitope and paratope regions. Model 1 (blue), model 3 (orange) and model 5 (green) were generated using Vakser Labs GRAMM docking web application and the contact results were analyzed using UCSF Chimera.

The graphs above can be used to draw conclusions of what interactions are occurring between the epitope and paratope regions of the proteins. Non-polar amino acids are hydrophobic so there are minimal hydrophobic interactions occurring. The main interactions are occurring from Polar uncharged amino acids which may form strong hydrogen bonding and electrostatic interactions between the desired regions. Overall, most contacts between all 3 models were between Glutamine 124 and Aspartate 148 showing there is a dominance of electrostatic interactions contributing to the bonding of the paratope and epitope regions.

Hydrophobic Interactions: Hydrophobic interactions can be inferred from regions in brown colouring, which indicate higher amounts of hydrophobicity. These pockets/regions tend to attract each other, forming interactions similar to how hydrophobic cores stabilize protein structures. Observing these regions in their involvement with binding between proteins can offer insights in hydrophobic forces. .

Electrostatic Interactions: Electrostatic interactions, represented by red (negative) and blue (positive) regions, highlight areas of attraction and repulsion. Where opposing charges are close in proximity, a strong attractive force is likely to occur. A repelling effect will occur when similar charges are in proximity to one another. These areas may hinder binding or promote conformational changes to avoid electrostatic repulsion. These regions can offer a deeper understanding of how proteins and other molecules might interact and the potential stability of such complexes.

4. Molecular Dynamics Simulations

GROMACS

We began the use of GROMACS for our molecular dynamics simulations when we discovered that employing Alliance (Digital Research Alliance of Canada) would not be feasible for logistical reasons. However, issues made themselves apparent with its use. Most importantly, the duration of time required to receive outputs from GROMACS was detrimental to our time-lin; although the software is optimized to be quick, its usage is reliant on the processing and graphical power of your personal computer, which not only restricted the quantity of results that could be obtained but also prolonged the time required to obtain results. To be more exact, these simulations span 1 to 2 nanoseconds; typically, we would aim for several hundred for an accurate result. To prepare for the use of Molecular dynamics simulations software we carried out cysteine contact analysis to determine locations of disulfide bridges as they can increase the stability of PbEL04. There are 22 total cysteines on PbEL04 and 15 disulfide bridges were found. This was done through Chimera. There were other significant issues; our waterbox was simply too small for our models. Albeit, by two angstroms, it was enough to cause a few atoms that reached the waterbox’s edge to jump to the opposite side, which interfered with the movie production and reduced accuracy of the visualized protein models. Additionally, our 40C simulation resulted in a very strange outcome. The protein appears to explode on the last frame of the simulation despite reacting regularly to the water over the last 1000 picoseconds. This caused massive irregularities on our analysis graphs. After our limited success with GROMACS, we contacted an advisor to enquire about the possibility of using a supercomputer. The supercomputer uses the AMBER software which employs different methodology and file nomenclature. We established a

10Å cubic waterbox and solvated to 150nM with Cl and Na ions, overall z pH at 7.4.

Values and Their Definitions

RMS-F : The RMSF value measures localized fluctuations within a system. Higher RMSF can indicate greater flexibility or motion in specific regions. Lower RMSF values can indicate more stable/rigid areas.

RMSD : This value shows to what degree the system deviates from its reference structure over time. Significant fluctuations may indicate conformational changes or instability in the system.

H-Bonds : Hydrogen bonds play a critical role in maintaining structural integrity and stability in biomolecules, consistent number of hydrogen bonds correlates with system stability.

Radius of Gyration (Rg): The Rg value measures the compactness of a structure. It provides insight into the overall shape and size of the molecule over time during a simulation. A decrease is indicative of folding/compactness while an increase indicates unfolding/expansion.

Solvent Accessible Surface Area (SASA) The SASA value measures the amount of surface area available to the solvent. A smaller value indicates a compactness and folding of the protein while a larger value indicates an expansion and greater surface exposure. SASA is important to infer stability

GROMACS, Analysis of PbEL04 at Different Temperatures

GROMACS 25C

Figure 3.05: RMS fluctuation of solvated PbEL04 at 25℃, 10Å cubic waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Residue number corresponds to X-axis. The Y-axis represents a fluctuation of distance (nm) of each residue over time.

Figure 3.1: RMSD fluctuation of solvated PbEL04 at 25℃, 10Å cubic waterbox and solvated to 150 nM with Cl and Na ions, overall pH at 7.4. RMSD value in nanometers is reflecting the Y-axis. Time in nanoseconds (ns) is representative of the X-axis.

Figure 3.15: Number of h-bonds of solvated PbEL04 at 25℃, 10Å cubic waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Time in picoseconds (ps) is representative of the X-axis.

Figure 3.2: Radius of gyration (Rg) of solvated PbEL04 at 25℃, 10Å cubic waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Nanometers are representative of the Y-axis. Time in picoseconds (ps) is representative of the X-axis.

Figure 3.25: Solvent accessible surface area (SASA) of solvated PbEL04 at 25℃, 10Å cubic waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Surface area (nm^2) is representative of the Y-axis and time is represented in picoseconds (ps) on the X-axis.

GROMACS 35C

Video 3.1: Molecular Dynamics simulation movie of PbEL04 showcasing atom jumps at 35℃ with a 10Å cubic waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Simulated through GROMACs and recorded through Pymol.

Figure 3.3: RMS fluctuation of solvated PbEL04 at 35℃, 10Å cubic waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Residue number corresponds to X-axis. The Y-axis represents a fluctuation of distance (nm) of each residue over time.

Figure 3.35: RMSD fluctuation of solvated PbEL04 at 35℃, 10Å cubic waterbox and solvated to 150 nM with Cl and Na ions, overall pH at 7.4. RMSD value in nanometers is reflecting the Y-axis. Time in nanoseconds (ns) is representative of the X-axis.

Figure 3.4: Number of h-bonds of solvated PbEL04 at 35℃, 10Å cubic waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Time in picoseconds (ps) is representative of the X-axis.

Figure 3.45: Radius of gyration (Rg) of solvated PbEL04 at 35℃, 10Å cubic waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Nanometers are representative of the Y-axis. Time in picoseconds (ps) is representative of the X-axis.

Figure 3.5: Solvent accessible surface area (SASA) of solvated PbEL04 at 35℃, 10Å cubic waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Surface area (nm^2) is representative of the Y-axis and time is represented in picoseconds (ps) on the X-axis.

GROMACS 40C

Figure 3.55: Screenshot of frame 112/113 of PbEL04 GROMACS 40C molecular dynamics simulation. Obtained through Pymol.

Figure 3.6: Screenshot of frame 113/113 of PbEL04 GROMACS 40C molecular dynamics simulation. Exploded protein. Obtained through Pymol.

Figure 3.65: RMS fluctuation of solvated PbEL04 at 40℃, 10Å cubic waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Residue number corresponds to X-axis. The Y-axis represents a fluctuation of distance (nm) of each residue over time.

Figure 3.7: RMSD fluctuation of solvated PbEL04 at 40℃, 10Å cubic waterbox and solvated to 150 nM with Cl and Na ions, overall pH at 7.4. RMSD value in nanometers is reflecting the Y-axis. Time in nanoseconds (ns) is representative of the X-axis. The graph shows an absurd climb at the end of the simulation.

Figure 3.75: Number of h-bonds of solvated PbEL04 at 40℃, 10Å cubic waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Time in picoseconds (ps) is representative of the X-axis.

Figure 3.8: Radius of gyration (Rg) of solvated PbEL04 at 40℃, 10Å cubic waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Nanometers are representative of the Y-axis. Time in picoseconds (ps) is representative of the X-axis. The drop is indicative of a crushed structure at the end, which is in line with the visual model at 40C.

Figure 3.85: Solvent accessible surface area (SASA) of solvated PbEL04 at 40℃, 10Å cubic waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Surface area (nm^2) is representative of the Y-axis and time is represented in picoseconds (ps) on the X-axis. The crash is indicative of an issue with the simulation.

Despite the fact that the GROMACS simulations have produced a great deal of work, very little can be deduced from the information and data provided. The simulations are too short to draw any conclusions and results. However, using GROMACS provided a solid foundational grasp of our molecular dynamics objectives. Most crucially, we needed simulations that extended to 200-300 nanoseconds to adequately come to a decision regarding the state of the protein’s stability; we learned that our values of our H-bonds, SASA, and RMSD on our graphs needed a progression of stability as the simulation was running. Through GROMACS we also learned that the size and shape of our waterbox was not as effective as it could have been, we increased our waterbox from 10Å to 12Å and changed from a cubic shape to an octahedral waterbox. The increase and change in shape helped avoid atom jumps in our AMBER simulations.

AMBER

AMBER was utilized when access to the supercomputer was granted. PbEL04 was solvated in an 12Å octahedral waterbox. Additionally, 150nM Na and Cl ion concentration was introduced. Overall, this system was modeled under conditions with a pH of 7.4; histidine residues were appropriately protonated using H++.

AMBER, Analysis of PbEL04 at Different Temperatures

AMBER 25C

Video 3.2: Molecular Dynamics simulation movie of PbEL04 at 25℃ with a 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Simulated through AMBER and recorded through Pymol.

Figure 4.05: RMS fluctuation of solvated PbEL04 at 25℃, 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Residue number corresponds to X-axis. The Y-axis represents a fluctuation of distance (Å) of each residue over time.

Figure 4.1: RMSD fluctuation of solvated PbEL04 at 25℃, 12Å octahedral waterbox and solvated to 150 nM with Cl and Na ions, overall pH at 7.4. RMSD value in angstroms (Å) is reflecting the Y-axis. Time in nanoseconds (ns) is representative of the X-axis.

Figure 4.15: Number of h-bonds of solvated PbEL04 at 25℃, 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Time in nanoseconds (ns) is representative of the X-axis.

Figure 4.2: Radius of gyration (Rg) of solvated PbEL04 at 25℃, 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Angstroms (Å) are representative of the Y-axis. Time in nanoseconds (ns) is representative of the X-axis.

Figure 4.25: Solvent accessible surface area (SASA) of solvated PbEL04 at 25℃, 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Surface area (nm^2) is representative of the Y-axis and time is represented in nanoseconds (ns) on the X-axis.

AMBER 35C

Video 3.3: Molecular Dynamics simulation movie of PbEL04 at 35℃ with a 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Simulated through AMBER and recorded through Pymol.

Figure 4.3: RMS fluctuation of solvated PbEL04 at 35℃, 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Residue number corresponds to X-axis. The Y-axis represents a fluctuation of distance (Å) of each residue over time.

Figure 4.35: RMSD fluctuation of solvated PbEL04 at 35℃, 12Å octahedral waterbox and solvated to 150 nM with Cl and Na ions, overall pH at 7.4. RMSD value in angstroms (Å) is reflecting the Y-axis. Time in nanoseconds (ns) is representative of the X-axis.

Figure 4.4: Number of h-bonds of solvated PbEL04 at 35℃, 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Time in nanoseconds (ns) is representative of the X-axis.

Figure 4.45: Radius of gyration (Rg) of solvated PbEL04 at 35℃, 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Angstroms (Å) are representative of the Y-axis. Time in nanoseconds (ns) is representative of the X-axis.

Figure 4.5: Solvent accessible surface area (SASA) of solvated PbEL04 at 35℃, 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Surface area (nm^2) is representative of the Y-axis and time is represented in nanoseconds (ns) on the X-axis.

AMBER 40C

Video 3.4: Molecular Dynamics simulation movie of PbEL04 at 40℃ with a 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Simulated through AMBER and recorded through Pymol.

Figure 4.55: RMS fluctuation of solvated PbEL04 at 40℃, 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Residue number corresponds to X-axis. The Y-axis represents a fluctuation of distance (Å) of each residue over time.

Figure 4.6: RMSD fluctuation of solvated PbEL04 at 40℃, 12Å octahedral waterbox and solvated to 150 nM with Cl and Na ions, overall pH at 7.4. RMSD value in angstroms (Å) is reflecting the Y-axis. Time in nanoseconds (ns) is representative of the X-axis.

Figure 4.65: Number of h-bonds of solvated PbEL04 at 40℃, 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Time in nanoseconds (ns) is representative of the X-axis.

Figure 4.7: Radius of gyration (Rg) of solvated PbEL04 at 35℃, 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Angstroms (Å) are representative of the Y-axis. Time in nanoseconds (ns) is representative of the X-axis.

Figure 4.75: Solvent accessible surface area (SASA) of solvated PbEL04 at 40℃, 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Surface area (nm^2) is representative of the Y-axis and time is represented in nanoseconds (ns) on the X-axis.

Comparison of different temperatures

Figure 4.8: RMS fluctuation of solvated PbEL04 across 3 different temperatures (25℃, 35℃, 40℃), 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Residue number corresponds to X-axis. The Y-axis represents a fluctuation of distance (Å) of each residue over time.

Figure 4.85: RMSD fluctuation of solvated PbEL04 across 3 different temperatures (25℃, 35℃, 40℃), 12Å octahedral waterbox and solvated to 150 nM with Cl and Na ions, overall pH at 7.4. RMSD value in angstroms (Å) is reflecting the Y-axis. Time in nanoseconds (ns) is representative of the X-axis.

Figure 4.9: Number of h-bonds of solvated PbEL04 across 3 different temperatures (25℃, 35℃, 40℃), 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Time in nanoseconds (ns) is representative of the X-axis. Color scheme: 25℃ – Blue, 35℃ – Orange, 40℃ – Green.

Figure 4.95: Radius of gyration (Rg) of solvated PbEL04 across 3 different temperatures (25℃, 35℃, 40℃), 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Angstroms (Å) are representative of the Y-axis. Time in nanoseconds (ns) is representative of the X-axis. Color scheme: 25℃ – Blue, 35℃ – Orange, 40℃ – Green.

Figure 5.0: Solvent accessible surface area (SASA) of solvated PbEL04 across 3 different temperatures (25℃, 35℃, 40℃), 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Surface area (nm^2) is representative of the Y-axis and time is represented in nanoseconds (ns) on the X-axis. Color scheme: 25℃ – Blue, 35℃ – Orange, 40℃ – Green.

AMBER, Analysis of PbEL04-Nanobody at Different Temperatures

Video 3.5: Molecular Dynamics simulation movie of PbEL04-Nanobody at 25℃ with a 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. PbEL04 and nanobody are coloured in blue and pink respectively. Simulated through AMBER and recorded through Pymol.

Figure 5.05: RMS fluctuation of solvated PbEL04-Nanobody structure at 25℃, 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. PbEL04 Residues. Residue number corresponds to X-axis. The Y-axis represents a fluctuation of distance (Å) of each residue over time

Figure 5.1: RMS fluctuation of solvated PbEL04-Nanobody structure at 25℃, 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Nanobody Residues. Residue number corresponds to X-axis. The Y-axis represents a fluctuation of distance (Å) of each residue over time

Figure 5.11: RMSD fluctuation of solvated PbEL04-Nanobody structure at 25℃, 12Å octahedral waterbox and solvated to 150 nM with Cl and Na ions, overall pH at 7.4. RMSD value in angstroms (Å) is reflecting the Y-axis. Time in nanoseconds (ns) is representative of the X-axis.

Figure 5.2: Number of h-bonds of solvated PbEL04-Nanobody structure at 25℃, 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Time in nanoseconds (ns) is representative of the X-axis.

Figure 5.25: Radius of gyration (Rg) of solvated PbEL04-Nanobody structure at 25℃, 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Angstroms (Å) are representative of the Y-axis. Time in nanoseconds (ns) is representative of the X-axis.

Figure 5.3: Solvent accessible surface area (SASA) of solvated PbEL04-Nanobody structure at 25℃, 12Å octahedral waterbox and solvated to 150nM with Cl and Na ions, overall pH at 7.4. Surface area (nm^2) is representative of the Y-axis and time is represented in nanoseconds (ns) on the X-axis.

According to the RMSD of pbEL04 across temperatures, the structure of pbEL04 finds conformational stability over time. This is implied by the fact that after 250 ns, the RMSDs of pbEL04 is no longer rampantly deviating. The number of hydrogen bonds for all three systems remain stable over the course of time which indicates that important hydrogen bonds are not being broken. The radius of gyration (RoG), generally speaking, measures the compactness of the biomolecule and, after ~250 ns, the RoG of the PbEL04 stabilizes which suggests that pbEL04’s is structurally stable. Furthermore, the solvent accessible surface area (SASA) also stabilizes and stops decreasing around ~250ns . Overall, all evidence points to pbEL04 being conformationally stable. However, that leaves only ~150ns of useful simulation where pbEL04 is stable. Thus, for future experiments, we want to increase our timescale to at least 1000 ns and up to 2000 ns. To determine whether this molecular behavior is reproducible we would also increase the amount of replicates to at least three. To see the limits at where pbEL04 becomes unstable, we would increase the range of our tested temperatures as well.