Difference between revisions of "Part:BBa K4347007:Design"
Victor5688 (Talk | contribs) (→Design Considerations) |
Victor5688 (Talk | contribs) |
||
| Line 34: | Line 34: | ||
===Design Considerations=== | ===Design Considerations=== | ||
| − | Since the goal of these point mutations was to increase thermal stability and not necessarily improve polymerase function, residues in the polymerase active site and fingers domain were avoided. In an article by | + | Since the goal of these point mutations was to increase thermal stability and not necessarily improve polymerase function, residues in the polymerase active site and fingers domain were avoided. In an article by Raghunathan & Marx [[Part:BBa_K4347007:Design#References|<sup>[3]</sup>]], it was found that only 25% of the mutations made in the fingers domain of Taq still resulted in a PCR active polymerase whereas over 70% and 60% of the mutations in the thumb and palm domains resulted in a PCR active polymerase. Due to the sequence similarity of Bst and Taq, these inactive mutations would likely have the same effect on Bst. It was further confirmed that residues 549, 582 and 584 in Bst were not involved with contacting the DNA strand and were not part of the polymerases active site[[Part:BBa_K4347007:Design#References|<sup>[4]</sup>]]. |
| − | |||
===Source=== | ===Source=== | ||
| Line 49: | Line 48: | ||
3. https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2007.05954.x | 3. https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2007.05954.x | ||
| + | |||
| + | 4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6231770/ | ||
Revision as of 19:43, 12 July 2022
Bst with point mutations for enhanced thermal stability codon optimized for E.coli
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 766
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Sequence alignment
Since Bst is structurally homologous to Klentaq polymerase, we fetched the Bst (6MU6) and Klentaq (6QV4) FASTA sequences from the Protien Data Bank and ran a multiple sequence alignment using Seaview. A good amount of conservation between the amino acid sequences was found between the two polymerases.
Residues to mutate
Since there is little literature about point mutations on Bst, point mutations made in Taq (Klentaq) were sought after. Three notable point mutations were found in the Klentaq thumb domain at positions K505A, K540A and K542A where each Lysine (K) residue was switched to an Arginine residue (R)[1]. After superimposing and aligning the structures and sequences in Pymol, the corresponding positions in Bst were found:
- K505 in Klentaq = K549 in Bst
- K540 in Klentaq = K582 in Bst
- K542 in Klentaq = Q584 in Bst
To confirm if these residues in Bst were suitable to mutate, the amino acid stability was estimated using a protien simulation software YASARA. A position scan was ran for each residue to measure the change in free energy (Kcal/mol) if the other 19 amino acid residues were mutated into that position. A mutation is classified as stabilizing if the change in free energy is ≤-1 kcal/mol, it is classified as destabilizing if the change is ≥1 kcal/mol, and neutral if it falls between these values [2]. The changes in free energy for mutations at residues K549, K582 and K584 are shown below:
Further research was conducted to narrow down on the most optimal amino acid mutation for each position. It was found that hydrophobic interactions play a role in protien thermal stability[3], thus substitutions of amino acids with hydrophobic side chains were made in accordance to the data obtained from the YASARA simulations:
- K549 --> W549 (-0.126 kcal/mol)
- K582 --> L582 (-1.632 kcal/mol)
- Q584 --> L584 (-2.039 kcal/mol)
To confirm if these point mutations were significant, the overall change in free energy was calculated and compared between the wildtype Bst and modified Bst. The overall change in free energy of wild-type Bst was calculated to be -150.13 kcal/mol, and the overall stability of the mutated Bst was calculated to be -151.81 kcal/mol, thus indicative of a more thermally stable protein.
Design Considerations
Since the goal of these point mutations was to increase thermal stability and not necessarily improve polymerase function, residues in the polymerase active site and fingers domain were avoided. In an article by Raghunathan & Marx [3], it was found that only 25% of the mutations made in the fingers domain of Taq still resulted in a PCR active polymerase whereas over 70% and 60% of the mutations in the thumb and palm domains resulted in a PCR active polymerase. Due to the sequence similarity of Bst and Taq, these inactive mutations would likely have the same effect on Bst. It was further confirmed that residues 549, 582 and 584 in Bst were not involved with contacting the DNA strand and were not part of the polymerases active site[4].
Source
PDB: 6MU5: https://www.rcsb.org/structure/6mu5
References
1. https://patents.google.com/patent/WO2009155464A2/en
2. https://www.frontiersin.org/articles/10.3389/fbioe.2020.558247/full
3. https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2007.05954.x