Difference between revisions of "Part:BBa K3843001:Design"

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===Design Notes===
 
===Design Notes===
  
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* * REPLACE ABOVE SEQUENCE WITH IMPROVED SEQUENCE * *
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To obtain the affinity-improved sequence, the original protein was first computationally mutated at every residue to every amino acid. Then, the stability of the mutated protein was assessed through overall energy scoring of the lone protein using Rosetta. Ultimately, only mutations occurring near the PEA-binding site were considered, as these mutations were most likely to affect binding. Next, the protein's binding affinity to phenylethylamine was assessed using ligand docking in AutoDock Vina as well as molecular dynamics simulations in GROMACS, which returned an energy score for the phenylethylamine+1UTM complex; lower energy scores indicated a higher binding affinity. The mutant with the highest binding affinity, where the mutation was in the active site of the protein, was chosen as the improved version of 1UTM. Combining multiple mutations by this process, the final affinity-improved 1UTM included the following mutations: Y204F, C195D, and S190T. The final protein was back-translated and codon optimized for <i>E. coli K12</i>. As well, illegal restriction sites for RFC[25] were removed to facilitate fusion protein construction.
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To obtain the affinity-improved sequence, the original protein was first computationally mutated at every residue to every amino acid. Then, the stability of the mutated protein was assessed through overall energy scoring of the lone protein using Rosetta. Ultimately, only mutations occurring near the PEA-binding site were considered, as these mutations were most likely to affect binding. Next, the protein's binding affinity to phenylethylamine was assessed using ligand docking in AutoDock Vina as well as molecular dynamics simulations in GROMACS, which returned an energy score for the phenylethylamine+1UTM complex; lower energy scores indicated a higher binding affinity. The mutant with the highest binding affinity, where the mutation was in the active site of the protein, was chosen as the improved version of 1UTM. Combining multiple mutations by this process, the final affinity-improved 1UTM included the following mutations: LIST MUTATIONS
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===Source===
 
===Source===

Latest revision as of 18:00, 19 October 2021


Affinity-improved PEA-binding salmon trypsin (1UTM+)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

To obtain the affinity-improved sequence, the original protein was first computationally mutated at every residue to every amino acid. Then, the stability of the mutated protein was assessed through overall energy scoring of the lone protein using Rosetta. Ultimately, only mutations occurring near the PEA-binding site were considered, as these mutations were most likely to affect binding. Next, the protein's binding affinity to phenylethylamine was assessed using ligand docking in AutoDock Vina as well as molecular dynamics simulations in GROMACS, which returned an energy score for the phenylethylamine+1UTM complex; lower energy scores indicated a higher binding affinity. The mutant with the highest binding affinity, where the mutation was in the active site of the protein, was chosen as the improved version of 1UTM. Combining multiple mutations by this process, the final affinity-improved 1UTM included the following mutations: Y204F, C195D, and S190T. The final protein was back-translated and codon optimized for E. coli K12. As well, illegal restriction sites for RFC[25] were removed to facilitate fusion protein construction.

Source

The original sequence of this part, used for computational rational protein design, was obtained from GenBank: https://www.ncbi.nlm.nih.gov/nuccore/X70071

With that said, the sequence of this part is a mutated version, obtained through computational rational protein design, as described above.

References

Leiros, H. K. S., Brandsdal, B. O., Andersen, O. A., Os, V., Leiros, I., Helland, R., Otlewski, J., Willassen, N. P., & Smalås, A. O. (2009, January 1). Trypsin specificity as elucidated by lie calculations, x‐ray structures, and association constant measurements. Protein Science, 13(4), 1056-1070. https://onlinelibrary.wiley.com/doi/full/10.1110/ps.03498604.

Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C. & Ferrin, T. E. UCSF Chimera--a visualization system for exploratory research and analysis (Version 1.15). J Comput Chem. 2004; 25(13): 1605-1612. https://www.ncbi.nlm.nih.gov/pubmed/15264254