Improving the Thermostability of T4 Lysozyme Using Protein FEP+ Guided Design

Tutorial Created with Software Release: 2025-3
Topics: Biologics Drug Discovery, Enzyme Engineering, Free Energy Perturbation (FEP)
Products Used: FEP+

Tutorial files

2.6 MB
This tutorial is written for use with a 3-button mouse with a scroll wheel.
Words found in the Glossary of Terms are shown like this: Workspacethe 3D display area in the center of the main window, where molecular structures are displayed

 

Tip: You can hover over a glossary term to display its definition. You can click on an image to expand it in the page.
Abstract:

 

In this tutorial, you will learn how to use Protein FEP+ to guide the design of protein mutants to improve thermostability. Visual inspection of the protein structures and residue scanning calculations will be insufficient to predict the effect of a particular mutation on the stability of a phage T4 lysozyme. Protein FEP+ on the contrary accurately reproduces the experimental estimate and helps finding a rationale for the underlying structural mechanism.

 

For a more technical introduction to setting up and validating Protein FEP+ models, see the Obtaining Protein Free Energy Perturbation Thermostability Predictions for Single Point Mutations tutorial.

Tutorial Content

  1. Introduction to Protein Thermostability Prediction

  1. Creating Projects and Importing Structures

  1. Visual Inspection of the Wildtype and Mutant

  1. Protein Thermostability Prediction with Protein FEP+

  2. Analyzing Wildtype and Mutant Structures

  1. Conclusion and References

  1. Glossary of Terms

1. Introduction to Protein Thermostability Prediction

When designing protein-based therapeutics or enzymes for industrial or consumer applications, it is crucial to understand whether an introduced mutation would adversely affect their thermostability, i.e. the ability of a protein to resist structural changes caused by heat. Experimental approaches to determine a change in thermostability due to a mutation can be expensive, slow and may sometimes not be feasible at all.

Computational or in silico mutagenesis, on the other hand, can be performed on any system, and there are different tools that can be used to predict thermostability. Most lack the ability to capture the dynamics of a protein system, as well as the effect of solvent molecules on the stability of a protein's folded state. One such method is residue scanning with Prime MM-GBSA (molecular mechanics with generalized Born and surface area), which provides qualitative results. Its high-throughput nature makes it an attractive tool for classifying a mutation as beneficial, deleterious, or unlikely to have a significant effect on thermostability, and so can be used to filter through large numbers of mutations.

Protein Free Energy Perturbation (FEP+) is a physics-based approach to the thermostability prediction task that uses explicit solvent, molecular dynamics, and a state-of-the-art force field (OPLS4) to compute both the enthalpic and entropic contributions to the free energy of a system. This gives accurate predictions, but at a higher computational cost. It can be afforded to be used for a selected number of mutations.

FEP+ residue scanning bridges the gap between the fast but approximate MM-GBSA and costly but accurate Protein FEP+, as it’s suited for screening larger numbers of mutations while still capturing the dynamics of the system. See the Identifying impactful mutations using FEP+ residue scanning tutorial for a guided introduction to applying this method for the prediction of binding affinities within a ternary complex.

In addition to prospectively identifying promising mutants, accurate in silico methods can help provide a rationale for the success or failure of specific mutations using computational structural analysis. In other words, they can be used to understand and explain the underlying structural mechanism, which can be difficult to achieve in experiments.

In this tutorial, you will investigate the influence of a single point mutation, where visual inspection and intuition, as well as the use of residue scanning, would be insufficient to predict whether the addition of a bulkier side chain at a particular position would improve thermostability. FEP+, on the contrary, provides accurate results and, together with computational structural analysis, helps to find an explanation for the influence of this specific mutation.

For a more in depth introduction to the technical details of protein thermostability predictions with protein FEP+, please see the Validating a Protein Free Energy Perturbation Model for Thermostability Predictions for Single Point Mutations tutorial. For more information on residue scanning, please refer to the MM-GBSA Residue Scanning documentation and the Improving Antibody Stability/Affinity Using MM-GBSA Residue Scanning tutorial.

2. Creating Projects and Importing Structures

At the start of the session, change the file path to your chosen Working Directorythe location where files are saved in BioLuminate to make file navigation easier. Each session in BioLuminate begins with a default Scratch Projecta temporary project in which work is not saved, closing a scratch project removes all current work and begins a new scratch project, which is not saved. A BioLuminate project stores all your data and has a .prj extension. A project may contain numerous entries corresponding to imported structures, as well as the output of modeling-related tasks. Once a project is created, the project is automatically saved each time a change is made.

Structures can be built in BioLuminate or can be imported from the PDB or your Working Directorythe location where files are saved using File > Import Structures, and are automatically added to the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion and Project Tabledisplays the contents of a project and is also an interface for performing operations on selected entries, viewing properties, and organizing structures and data. The Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion is located to the left of the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed. The Project Tabledisplays the contents of a project and is also an interface for performing operations on selected entries, viewing properties, and organizing structures and data can be accessed by Ctrl+T (Cmd+T) or Window > Project Table if you would like to see an expanded view of your project data.

OR

  1. Double-click the BioLuminate icon.
    • (No icon? See Starting BioLuminate)
    • This tutorial uses BioLuminate, but this workflow can be performed in Maestro or BioLuminate. Use whichever interface you are comfortable with or typically use for your projects.

Figure 2-1. Changing the Working Directory via the option in the main window.

  1. Go to File > Change Working Directory.
  2. Browse to the directory you want to use as your Working Directorythe location where files are saved, and click Select Folder.
  3. Pre-generated input and result files are included for running jobs or examining output. Download the .zip file here: schrodinger.com/sites/default/files/s3/release/current/Tutorials/zip/fep_protein_thermostability.zip.
  4. After downloading the .zip file, unzip the contents in your Working Directorythe location where files are saved for ease of access throughout the tutorial.

Figure 2-2. Saving the project via the Save Project As dialog box.

  1. Go to File > Save Project As.
  2. Change the File name to 1L63_cavity, click Save.
    • The project is now named FEP_Stability_1L63.prj and is saved in your Working Directorythe location where files are saved.

Figure 2-3. Importing the prepared structure of the protein.

  1. Go to File > Import Structures….
    • The Import dialog box opens.
  2. Navigate to your Working Directorythe location where files are saved, select the file 1L63_prepared.mae and click Open. Confirm by clicking Import.
    • A banner appears and a group is added to your Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.

3. Prerequisites for Protein FEP+ and System Preparation

Structures obtained from the PDB, vendors, and other sources often lack the necessary information

for performing modeling-related tasks. Typically, these files are missing hydrogens, partial charges,

side chains, and/or whole loop regions. In order to make these structures suitable for modeling

tasks, we use the Protein Preparation Workflow to resolve issues.

In this tutorial, the 1L63 protein structure was retrieved from the PDB and prepared following the method described in this publication. However, these preparation steps are a necessary part of the process and must be done before any FEP+ calculations. Please see the Introduction to Structure Preparation and Visualization tutorial for instructions on using the Protein Preparation Workflow and Preparing Protein and Ligand Structures for FEP+ and the Protein FEP+ Best Practices for tips on structure preparation for FEP+.

In addition to the protein structure, you should have set up a Protein FEP+ model and validated on a small set of mutants whose thermostability relative to the wild type is known experimentally. Optimally, these mutants should span a large range of ∆∆G compared to the wild type. We recommend only using Protein FEP+ models prospectively whose prediction error on the validation set does not exceed 1.3 kcal/mol. See the Validating a Protein Free Energy Perturbation Model for Thermostability Predictions for Single Point Mutations tutorial for how FEP+ validation was performed for this system.

3. Inspecting a Promising Mutation Site

There is no clear and established procedure for determining promising mutation sites. Ideas can come from experimental observations, computational analysis, chemical intuition and/or serendipity. For the purposes of this tutorial, let’s say that previous investigation has yielded the serine in position 117 of the phage T4 lysozyme as a promising candidate for creating more thermally stable mutants. Before proceeding to generate the mutants, let’s first have a look at the environment of this residue. This can give us an understanding of its role in the hydrogen bond network and how densely packed the protein is in that region.

Figure 3-1. Applying the BioLuminate Default presets for visualization.

  1. Go to Presets and select BioLuminate Default.
    • The structure is now rendered in green with only the protein-ribbons and ions shown.

Figure 3-2. Selecting SER 117 and expanding the selection by a radius of  3Å.

 

First, you can identify the residue of interest in the workspace along with its immediate environment.

  1. In the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed Sequence Viewer, scroll to the right and select the residue SER 117 by left-clicking on the corresponding one-letter code (S).
    • SER 117 is selected in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed but not displayed.
  2. In the top toolbar, click Expand and select an expansion radius of 3Å.
    • The selection in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed is expanded to all residues within 3Å of SER 117, but all selected atoms are still not displayed.

Figure 3-3. Displaying the selected atoms and rendering them in a ball-and-stick representation.

Feel free to adjust the visualization to your preference. Here, we will use the ball-and-stick representation.

  1. Go to Style and display the selected atoms and render them in a ball-and-stick representation.
    • SER 117 and the surrounding residues are now displayed in a ball-and-stick representation in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

Figure 3-4. Displaying all interactions and contacts in the workspace.

By default, only ligand-receptor interactions are shown in the workspace, so to visualize the H-bond network within the receptor you can change the interaction settings.

  1. Right-click on the Interactions Toggle and select All for Non-covalent bonds and Pi interactions and Contacts/Clashes.
    • Interactions are shown in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

Figure 3-5. Interactions and clashes of SER 117 after quick minimization.

Now, you can inspect the region of interest.

  1. Fit the view to SER 117 and the surrounding residues, e.g. by clicking Fit view to selected atoms in the toolbar at the top.
  2. Have a look at the interactions and contacts between SER 117 and other residues.

While the backbone of SER 117 is involved in forming an alpha helix, the side chain of SER 117 forms a hydrogen bond to ASN 132. Mutating Ser 117 to an amino acid without a H-bond donor would break this hydrogen bond, which could negatively impact the protein thermostability, as this bond stabilizes the arrangement of two alpha-helices. It is however impossible to judge whether this would actually impact the overall thermostability from visualization alone.

 

Another question that stands to reason is whether this region will tolerate bulkier amino acid residues. Answering this question rigorously is again impossible when only working with this static structure, as proteins can be more flexible than superficially visible. Still, you can do a brief check by mutating the serine to something bulkier, like a phenylalanine.

Figure 3-6. Duplicating and renaming the entry.

We recommend always duplicating the structure you are working on before mutating or making edits using the 3D builder.

  1. Make a copy of your entry in the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion by right-clicking on the entry and selecting Duplicate > In Place.
    • A new entry of the same name is added to your Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
  2. Rename the new entry to 1L63_prepared_S117F.

Figure 3-7. Manual mutation of Ser 117 to Phe.

  1. Includethe entry is represented in the Workspace, the circle in the In column is blue the new entry in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed and right-click on any atom of SER 117, then select Mutate Residue and choose PHE.
    • The residue at position 117 is mutated from Ser to Phe.

Figure 3-8. Interactions and clashes of Phe 117 after a quick minimization.

The Phenylalanine you just added clashes badly with the surrounding protein, as its conformation was chosen based on typical bond and torsion angles of phenylalanines in protein structures rather than its specific environment. Sometimes, simple minimization of the residue and its surroundings can be sufficient to resolve clashes.

  1. Select the new PHE 117 residue.
  2. In the top toolbar, click Expand and select an expansion radius of 3Å again.
  3. Press Ctrl (Cmd) + M to minimize the selected atoms.

The phenylalanine residue seems to barely be able to fit between the two helices, but shows a lot of clashes with the environment. The former hydrogen bond to residue Asn 132 is broken, and the minimization to accommodate the bulkier side chain has slightly wedged the two alpha helices apart.

To summarize the insights from the quick inspection you just performed: The side chain of SER 117 forms a hydrogen bond that seems to stabilize the arrangement of two alpha helices, which might be important for the protein’s thermostability. Also, the environment at this residue position seems like it could be flexible enough to tolerate bulkier side chains. From minimization alone, it seems unlikely that something like a phenylalanine would fit. More concrete statements are not possible based on the rudimentary analysis we used.

4. Identifying promising mutants for investigation with FEP+

As mentioned in the introduction, the computational cost of FEP+ means that it is not feasible to screen large numbers of mutants with it directly. Instead, we recommend using MM-GBSA residue scanning and/or Protein FEP+ residue scanning as pre-filters to identify mutants to investigate with FEP+. For in-depth hands-on examples on using these methods, see the Identifying impactful mutations using FEP+ residue scanning tutorial.

For this tutorial, we have run these calculations for you and will only briefly discuss the results before using Protein FEP+ on a particularly promising mutation.

The MM-GBSA residue scanning results predict all residues as destabilizing, in particular the S117F mutation discussed above. Note that this estimate of the stability is essentially based on potential energies and is therefore only an approximation of the free energy.

This result is not surprising, as the method does not take into account the dynamics of the system, so side chain rearrangements in the region of the mutation to accommodate the bulkier phenylalanine side chain are not possible. An option can be used that allows some limited and local rearrangements and may slightly improve the results.

Residue scanning is commonly used to rank mutations by stability (ΔE) to select only the most promising mutations for further analysis with more expensive and accurate methods like FEP+, which does account for the dynamics of the system. In this case the S117F mutation would likely not be chosen for further investigation. Learn more about residue scanning with Prime MM-GBSA.

4.1 Setting up the FEP+ Calculation

In this section, you will learn how to set up and run a Protein FEP+ job with the FEP Protein Mutation panel to calculate the difference in thermostability due to a single point mutation.

As noted in the Protein FEP+ Best Practices, there are several specific recommendations for preparing a protein structure that will serve as an input for Protein FEP+ calculations. As the structure of the protein used in this tutorial is already prepared, these steps can be skipped here, but keep them in mind when applying the method in your own projects.

Figure 4-1. Loading the structure into the FEP Protein Mutation panel.

  1. Includethe entry is represented in the Workspace, the circle in the In column is blue and select(1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entry List (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries the entry 1L63_prepared (the wild type).
  2. Go to Tasks > Browse All > FEP+ > Protein FEP.
    • The FEP Protein Mutation panel opens.
  3. For Use structures from select Project Table (1 selected entry) and click Load.
    • The structure is loaded into the panel.
  4. For Calculation type select Stability.

Figure 4-2. Selecting the S117F single mutation.

For this tutorial, we will only analyze a single mutation – the S117F mutant briefly discussed above.

  1. In the Select residues in the table to mutate section, in the Single Mutations tab, scroll down to locate A:117(Ser).
  2. Click into the right column on Click to edit…, select PHE and click Close.
    • The panel updates the number of residues and mutants.

Figure 4-3. The job settings for the FEP run.

  1. Change the Job name to fep_prot_mutation_1L63_S117F.
  2. Optional: Click the cog.
    • The FEP Protein Mutation - Job Settings dialog box opens.
  3. Optional: Select a CPU Host for the Master Job and a Linux-based GPU Host for the Subjob(s), and specify the number of subjobs and GPUs.
  4. Click OK.

The Protein FEP job is now ready to run. Because the simulation utilizes Desmond and FEP+, the job requires a Linux-based host with GPUs. For this reason, and to save time, you do not need to run the actual calculation. Pre-generated result files for a run of the system can be found in the zip file you downloaded at the beginning. Feel free to run the simulation yourself, if you have access to suitable hardware.

More options for the job can be found in the FEP Advanced Options dialog box, which is accessible via the cog.

4.2 Analyzing the Results

In this section, you will load pre-generated Protein FEP+ results with the FEP+ panel and analyze the influence of the introduced mutation on the proteins thermostability compared to the experiment. You will look at the interactions of wildtype and mutant in the FEP+ – Analysis panel.

Please note that calculations may have been performed with an earlier version of the software and the results may not be exactly the same as those you produced in this tutorial. Especially for methods with a statistical component, such as MD simulations, an exact reproduction of a trajectory and its properties is only possible if the same random seed, software and hardware configuration are used. With sufficient sampling, all properties of the ensemble should be reproducible, while the properties of individual frames and the exact course of a trajectory may differ.

Figure 4-4. Loading the pre-generated results in the FEP+ panel.

  1. Go to Tasks > Browse All > FEP+ > FEP+.
    • The FEP+ setup panel opens.
  2. For Import structures or perturbation map from select File.
  3. Click Browse and navigate to your extracted zip file, select the file fep_prot_mutation_1L63_S117F_out.fmp.
    • The output is detected.
  4. Click Next.
    • The results are loaded and the Review panel opens in the overview.
    • The wildtype (WT) and mutant (A-SER117PHE) are added to the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion as a group.

Figure 4-5. Comparing the experimental and FEP+ result for the difference in thermostability.

You can already see the predicted ∆∆G of -1.1 kcal/mol between the wild type and the mutant. This closely matches the experimental reference value of -1.1 kcal/mol, which you should now add to the panel so quality metrics can be calculated.

  1. Double-click the N/A entry in the column Exp. Stability for the mutant (line two) and enter the experimental difference in stability of -1.1 kcal/mol.

Note: When working with multiple mutations, you can also import reference stability data from a file using the Affinity button in the FEP+ panel’s toolbar.

The FEP+ result reproduces well the experimental estimate of the difference in thermostability between the wildtype and the S117F mutant. The mutation actually increases the thermostability of the protein, contrary to the visual inspection and residue scanning discussed in the previous section. You can now investigate the reasons for this finding.

Figure 4-6. Opening the edge analysis.

The Analysis tab collects various quality metrics for the simulation (‘edge’) associated with each mutation. The quality metrics all look good, so you can dive into the Edge Analysis to understand which factors contribute to the ∆∆G.

 

  1. Switch to the Analysis tab.
  2. Click View… to open the Edge Analysis.
    • The FEP+ – Analysis panel opens.

 

Note: See the documentation for more details on interpreting the FEP+ quality metrics.

Figure 4-7. The interactions of residue 117 with other residues in the wildtype and the mutant.

You can view the interactions of various surrounding residues with the mutated site to understand how the mutation affects the local environment.

  1. Switch to the Residue Interactions tab, to investigate the interactions of residue 117 with other residues in the wildtype and the mutant.

Note: The term “Ligand” in this panel refers to the mutated residue (SER/PHE 117) and its immediate neighbors ASN 116 and LEU 118.

In the chart, there are two bars for each residue near the mutated site. The left-hand, solid bar shows the presence of interactions of the residue to the mutated site and its immediate neighbors (labeled here as “ligand”). The y-axis is given in % of simulation frames in which any atom of the residue interacts with any atom in the “ligand”, so the number can add up to more than 100% when multiple interactions are possible (e.g. H-bond interactions to both the backbone and sidechain of ARG 119).

Comparing the interactions of residue 117 in the wildtype (Ser 117) and the mutant (Phe 117), hydrogen bonds are mostly retained. A significant difference is the absence of the hydrogen bond between Phe 117 and Asn 132 that you saw in the visual inspection earlier. This interaction is broken as expected. However, there are two new and strong hydrophobic interactions in the mutant that are not present in the wildtype. Phe 117 interacts with Leu 133 and Phe 153 for approximately 80% of the simulation time. This explains the gain of negative free energy and thus the improved thermostability for the mutant compared to the wildtype on an energetic level.

The other tabs in the Edge Analysis panel provide additional information about the convergence and sampling for this simulation, which may be helpful when troubleshooting your own FEP+ jobs.

5. Analyzing Wild Type and Mutant Structures

In this section, you will investigate the structure of the wildtype and mutant proteins, to find an explanation for the improvement of thermostability due to the mutation.

Figure 5-1. Changing the visualization state of wildtype and mutant.

  1. Includethe entry is represented in the Workspace, the circle in the In column is blue both the WT and mutant entries from the FEP+ group in the workspace.
  2. Go to Presets and select BioLuminate Default.
  3. In the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed Sequence Viewer, select the residues Ser 117 and Phe 117 by left-clicking on the corresponding one-letter codes (S and F), while holding Ctrl (Cmd).
  4. Go to Style and display the selected atoms and render them in a ball-and-stick representation.

Figure 5-2. Creating a solid surface for the proteins.

  1. Select the protein from the Quick Select section (P) in the toolbar at the top.
  2. Go to Style and click on Surface.
    • The proteins are rendered in a solid surface in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

Figure 5-3. Rendering the protein surfaces in a see-through mesh.

  1. Click on the S behind the entry in the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion to open the workflow action menu and select Manage….
    • The Manage Surfaces panel opens.
  2. Select both surfaces (Ctrl/Cmd click) and  go to Display Options… and select Mesh as the Style. Confirm with OK and close the Manage Surfaces panel.
    • The surface is now displayed as a see-through mesh.
  3. Inspect both protein surfaces with special emphasis on the surroundings of residue 117.

Figure 5-4. The wildtype (left) and the mutant (right) protein rendered in a mesh surface.

There is a small cavity buried within the protein structure of the wildtype protein, next to Ser 117, which faces away from the cavity. In the mutant, the residue Phe 117 occupies exactly the space of the cavity in the wildtype, there is no cavity in the mutant protein.

Protein FEP+, in contrast to residue scanning, is able to describe the dynamics that are necessary for the surrounding residues to reorganize in such a way that a residue with a bulkier hydrophobic side chain can be accommodated. This fills the cavity (cavity-filling mutation) and leads to additional strong hydrophobic interactions within the protein, improving the thermostability of the protein.

Using cavity-filling mutations as a general approach to improve protein thermostability is not always successful. The L133F mutant was experimentally found to be less stable than the wildtype. Although it fills the cavity, it leads to unfavorable close contacts because the surrounding residues cannot adapt well enough to the structural change.

6. Conclusion and References

In this tutorial, you learned how to set up and run Protein FEP+ jobs to predict the change in thermostability between wildtype and mutated proteins. First, you visually inspected the structures of a phage T4 lysozyme wildtype and S117F mutant. Both intuition and residue scanning results suggested a deleterious effect of the mutation on the thermostability of the protein. However, Protein FEP+ was able to accurately predict this mutation to improve the thermostability, reproducing the experimental estimate of the change in free energy. Moreover, it unraveled an explanation for the effect of the mutation. Introducing a residue with a bulkier side chain fills a cavity within the protein (cavity-filling mutation) and leads to additional strong hydrophobic interactions with surrounding residues.

7. Glossary of Terms

Entry List - a simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion

Included - the entry is represented in the Workspace, the circle in the In column is blue

Project Table - displays the contents of a project and is also an interface for performing operations on selected entries, viewing properties, and organizing structures and data

Recent actions - This is a list of your recent actions, which you can use to reopen a panel, displayed below the Browse row. (Right-click to delete.)

Scratch Project - a temporary project in which work is not saved, closing a scratch project removes all current work and begins a new scratch project

Selected - (1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entry List (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries

Working Directory - the location where files are saved

Workspace - the 3D display area in the center of the main window, where molecular structures are displayed