Protein stability and dynamics

Protein stability

ProThermhttps://web.iitm.ac.in/bioinfo2/prothermdb/index.html FireProtDBhttps://loschmidt.chemi.muni.cz/fireprotdb/

ProTherm database

  • Compare the stabilities of the Adenylate kinase protein from different organisms based on data from the ProTherm database
    • Browse - Search → Protein: Adenylate kinase; Measure: CD
    • In Display Options, saelect items: ENTRY, PROTEIN, MUTATION (UniProt and PDB)
    • In Thermodynamics parameters, select: Tm, ΔTm
  • Why is the stability (Tm - melting temperature) of these proteins different?

FireProtDB database

  • Use the FireProtDB advanced search to find mutations that reduce the stability of the Adenylate kinase enzyme (Uniprot ID: P69441)
    • Search - Advanced → Uniprot ID: P69441 AND ΔΔG > 0 → Search
    • How many neutral and destabilizing mutations did you find?

Protein dynamics

RCSB PDBhttps://www.rcsb.org

NMR

  • In PyMOL, study the dynamics of the protein Rubredoxin as described by its NMR structure
    • Fetch the NMR structure 1BFY
    • Play the animation: click the "play" (►) button
    • Change the speed: Movie → Legacy movie maker → Frame rate → 5 FPS
    • Change visualization: Show → Lines. Note how the motions of the atoms are coordinated
    • Visualize the entire ensemble of structures: Movie → Legacy movie maker → Show all states. Note how some regions are overlapping more and others are more fuzzy: the latter have higher RMSD, indicating they are more flexible.

X-ray

  • In PyMOL, study the flexibility of the Rubredoxin protein as described by the X-ray structure
    • Fetch the X-ray structure 1IRN
    • Show anisotropic B-factors of atoms: type in the command-line: show ellipsoids, 1IRN
    • Remove waters: Action → remove waters
    • Which atoms have the highest B-factors?
  • Compare the dynamical information present in this structure with that from NMR
    • Superimpose the two structures (place them on top of each other): for model 1IRN click: Action → align → all to this (*/CA)
    • Hide hydrogen atoms: Hide → hydrogens → all
    • Visualize the X-ray structure using B-factor putty visualization: Action → preset → B-factor putty
    • Which regions show the highest flexibility for the two structures?
    • Explore the flexibility of residues LYS2, TYR13, PRO34 and LEU41: type sele resid 2+13+34+41
    • For the new model “sele” click: Show → sticks; for 1BFY click: Hide → cartoon
    • Observe the distribution of those residues in space for the NMR structure. Which ones are more flexible? Which ones are less flexible?

Dynamics databases

ProMode-Elastichttps://pdbj.org/promode-elastic ATLAShttps://www.dsimb.inserm.fr/ATLAS

ProMode-Elastic database

  • In the ProMode-Elastic database, study the Normal modes of the enzyme haloalkane dehalogenase DhaA:
    • Search for: 1CQW; click on “1cqw”
    • View the images of the displacement vectors for different modes: in the Interactive 3D Visualization panel, select: Mode: → Mode 1 / Mode 2 / Mode 3 / etc.
    • Visualize the animation of Mode 6: select: Mode: → Mode 6 → Visualization: → All Displacement Vectors / Animation

ATLAS database

  • In the ATLAS database, investigate MD simulations for the Orthoflavivirus capsid protein (UniProt ID: Q9Q6P4)
    • Click on search. Enter the UniProt ID: Q9Q6P4; down below, click on the entry “4oieA”
    • Check the flexibility: root-mean square fluctuation (RMSF) and B-factors vs. sequence. Which are the most flexible regions?
    • Check the MD evolution and convergence for the 3 replicas: RMSD vs. time. They are not exactly the same. Why is that?
    • Visualize the MD trajectory (the molecular motions) in the 3D Viewer: click Dynamics → Select animation (play button) → Start
    • Select a few residues in the sequence: these will be displayed in detail in the 3D viewer. Observe how they move in coordination.

MD calculation and analysis

BioBB web server:https://mmb.irbbarcelona.org/biobb-wfs

Calculation of MD simulations

  • Obtain the structure of the Orthoflavivirus capsid protein from the previous exercise
  • Prepare and run MD simulation with the BioBB web server
    • In the server, click: Create project → From Structure
    • Provide the structure: click: Select file → (find the 4oie.pdb file you just downloaded) → Submit
    • After a structure check, several warnings are listed. In this case, they can be handled automatically. Click: Next to summary. In the next page, click: Next to settings
    • Specify: force field, solvent model, box size, box type, ion concentration: keep the defaults
    • Check the box “Click to prepare configuration files”; you may change several MD settings, including the temperature (in K); click: Summary and launch the project; in the next page, click: Launch project.
    • Unfortunately, without access to a high performance computer (HPC), you can only run up to 500 ps.
    • The calculation will take several hours to finish.

Analysis of MD simulations

  • Analyze the results from the previous MD simulation in BioBB web server
    • Open pre-calculated results from the BioBB web server. Expand the “Analysis Results” tab. Visualize the initial structure and the motions during the MD; check the evolution of several properties in time: the energies of the system, radius of gyration, and very importantly, the RMSD.
    • Expand the “Project actions” tab. Click “Download results” to obtain the MD files. Unzip the downloaded file.
  • Visualize the MD simulation in PyMOL
    • In PyMOL, open the biobb.MDsetup.top.pdb file from the previous folder; open the biobb.MDsetup.xtc file; select Molecular object: biobb.MDsetup.top; click: Load.
    • Click: Show → lines
    • Play the MD animation: click the “play” (►) button. You can visualize all the protein atoms, waters and ions as they were simulated for 500 ps.
    • Click: Hide → Waters; Hide → spheres. Now you see only the motions of the protein atoms.
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