Engineering of protein structures I

Annotation of mutated residues

HotSpot Wizardhttps://loschmidt.chemi.muni.cz/hotspotwizard/
  • study the structure of wild-type haloalkane dehalogenase DhaA (PDB-ID 4E46) using HotSpot Wizard 3.0
    • set the minimum width of the tunnels to 1.0 Å: Advanced setting -> Calculation of tunnels -> Minimal probe radius
    • precalculated results: HSW-1, HSW-2, HSW-3, HSW-4, HSW-5
  • in which positions can we expect an undesirable effect on function and stability, and when can a rather neutral effect be expected?
  • which mutations are potential stability hot spots in terms of flexibility and evolution?
  • which mutations are most evolutionarily correlated?

Structure preparation of mutant proteins

PyMOL Mutagenesis

  • in PyMOL prepare the structure of the DhaA (4E46) protein carrying the I135F mutation
    • first, remove waters from the structure
    • Wizard → Mutagenesis → Protein
    • choose the best-fitting phenylalanine rotamer that will result in the least steric hindrance
ESMFold and PymolFoldhttps://github.com/JinyuanSun/PymolFold

ESMFold via PymolFold

  • use the PymolFold plugin for PyMOL to prepare the structure of the DhaA protein carrying the I135F mutation, and then see the mutation's confidence score (plddt)
    • In PyMOL command line:
      run https://raw.githubusercontent.com/JinyuanSun/PymolFold/main/pf_plugin.py
      run https://raw.githubusercontent.com/JinyuanSun/PymolFold/py27/predict_structure.py
    • Predict the single-point mutant:
      esmfold MSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYLWRNIIPHVAPSHRCIA PDLIGMGKSDKPDLDYFFDDHVRYLDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIACMEFIR PFPTWDEWPEFARETFQAFRTADVGRELIIDQNAFIEGALPKCVVRPLTEVEMDHYREPFLKPVDREPLW RFPNELPIAGEPANIVALVEAYMNWLHQSPVPKLLFWGTPGVLIPPAEAARLAESLPNCKTVDIGPGLHY LQEDNPDLIGSEIARWLPALHHHHHH

      color_plddt

    Comparison of the results

    • compare your constructed mutant structures with the actual crystal structure of the mutant (PDB-ID 3G9X)
    • focus not only on the mutated residue but also on its surroundings

    Prediction of mutation effect on protein stability

    FireProthttps://loschmidt.chemi.muni.cz/fireprotweb/

    FireProt

    • use PyMOL to hypothesize the effect of the following mutations on the stability of DhaA (PDB-ID 4E46)
      • all possible mutations of N41, D106, W107, and R179
      • mutations I135F and C176Y
      • verify your assumptions using FireProt 2.0 webserver
      • calculation setup:
        • click the STRUCTURE button, enter the PDB ID, and click the Process structure button
        • fill in the job title and your email (recommended - calculation takes a while)
        • click the Next button
        • you will be asked if you want to go to precalculated results - click Cancel
        • click the USER-DEFINED button (we are interested in specific mutation)
        • select the mutations of interest: click on a residue, then either select a specific mutation or all possible variants by clicking the Select all button
        • continue until all mutations are specified in the gray table at the bottom
        • click the Next button
        • click the Next button again without selecting any essential residues
        • click OK in the next dialog window
        • Do not submit the job - use the precalculated results
      • are the mutations predominantly stabilizing or destabilizing? And why?
      FireProtASRhttps://loschmidt.chemi.muni.cz/fireprotasr/

      FireProtASR

      • when stabilizing a protein, it is worth looking in its past, when there were harsh conditions
      • for this, we have a method called Ancestral protein reconstruction (ASR), which uses homologous sequences with the same active site to construct their parental proteins
      • explore the ancestors of DhaA using FireProtASR webserver
      • calculation setup:
        • click the Load example button, click the Validate button
        • fill in the job title and your email (recommended - calculation takes a while)
        • tick the box to agree with the academic license (under email)
        • click the Next button
        • you will be asked if you want to go to precalculated results - click Calculation settings
        • click the USER-DEFINED button (we are interested in specific mutation)
        • wait for essential residues to load from SwissProt
        • you can choose a one-step or two-step run
        • Do not submit the job - use the precalculated phylogenetic tree
        • explore the phylogenetic tree of DhaA
        • we're in the 2-step "expert mode", you can adjust the tree by clicking on the nodes
        • Do not submit the job - use the precalculated results
        • click the Show results button
        • in the top window, you can see the structure of DhaA, but more importantly, at the bottom, there is the phylogenetic tree with constructed ancestral sequences
      • explore the ancestors of DhaA by clicking on their respective nodes. Find its closest and oldest ancestors. Which will have more mutations? Which will be more stable? Will they differ from DhaA in function?
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