Structure-based mutation analysis (Phenylketonuria)
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Contents
Summary
In Task 8 the sequence of PAH was used for finding mutational effects, now the structure will be taken for these analysis. But how to find out, if a mutation changes the structure? Therefore, one calculates the energy of all atoms for the wildtype and the mutated structure and compares the results for changes. There are two different methods for this calculations given: Quantum Mechanics (QM) and Molecular Mechanics (MM). In QM the energy of all electrons in a protein is calculated. It is one of the most accurated methods, but it is very time consuming. In MM the energy of a system is calculated as a function of nuclear positions. It is very fast and easy to calculate, but it ignores electronic motions and is not as accurate as QM. Since QM is too time intensive and the results of MM are nearly as good as the ones calculated with QM, we use MM for the further analysis. Molecular Mechanics uses force fields for the energy calculation, which is defined as a sum of terms. The terms are non-bonded (electrostatic and Van-der-Waals) and bonded (Bond stretching, Angle stretching, bond rotation) interactions. For the structure based mutation analysis the SCWRL and FoldX webserver were used.
Structure selection
In some Tasks before, we used the protein structure of 2PAH as reference, but now we have to check some more constraints for the protein structure selection:
- Structure with the highest resolution (small Å value)
- Smallest R-factor
- Highest coverage
- pH-value ideally near physiological pH of 7.4
- No gaps (missing residues) included in the structure, so a consecutive numbering of residues should be given
To check which protein structure to use for further analysis, we compared the constraint data for all sequences given in the PAH (P00439) Uniprot entry. <figtable id="pro-struc">
| Protein | Method | Resolution(Å) | R-factor | pH | Gaps | Chain | Positions | Coverage % |
|---|---|---|---|---|---|---|---|---|
| 1DMW | X-ray | 2.00 | 0.20 | 6.80 | - | A | 118-424 | 67,92 |
| 1J8T | X-ray | 1.70 | 0.20 | 6.80 | - | A | 103-427 | 71.90 |
| 1J8U | X-ray | 1.50 | 0.16 | 6.80 | - | A | 103-427 | 71.90 |
| 1KW0 | X-ray | 2.50 | 0.22 | 6.80 | - | A | 103-427 | 71.90 |
| 1LRM | X-ray | 2.10 | 0.21 | 6.80 | - | A | 103-427 | 71.90 |
| 1MMK | X-ray | 2.00 | 0.20 | 6.80 | - | A | 103-427 | 71.90 |
| 1MMT | X-ray | 2.00 | 0.21 | 6.80 | - | A | 103-427 | 71.90 |
| 1PAH | X-ray | 2.00 | 0.18 | 6.80 | - | A | 117-424 | 68.14 |
| 1TDW | X-ray | 2.10 | 0.21 | 6.80 | - | A | 117-424 | 68.14 |
| 1TG2 | X-ray | 2.20 | 0.21 | 6.80 | - | A | 117-424 | 68.14 |
| 2PAH | X-ray | 3.10 | 0.25 | 7.00 | 136LEU-143ASP | A/B | 118-452 | 74.12 |
| 3PAH | X-ray | 2.00 | 0.18 | 6.80 | - | A | 117-424 | 68.14 |
| 1ANP | X-ray | 2.11 | 0.20 | 6.80 | - | A | 104-427 | 68.81 |
| 4PAH | X-ray | 2.00 | 0.17 | 6.80 | - | A | 117-424 | 68.14 |
| 5PAH | X-ray | 2.10 | 0.16 | 6.80 | - | A | 117-424 | 68.14 |
| 6PAH | X-ray | 2.15 | 0.17 | 6.80 | - | A | 117-424 | 68.14 |
</figtable> All proteins were found with the X-ray diffraction method. In <xr id="pro-struc"/> we can see, that the structure of 1J8U has a better resolution value as well as R-factor than the other structures. Although 2PAH has a better pH-value, a higher coverage and even two domains however the structure includes one gap. For this reason as well as the better R-factor and higher resolution value we have chosen the structure of 1J8U for the further analysis. Moreover, the structure includes the second highest coverage and also a very good pH-value.
Visualisation of used mutations
Following five mutations are mapped to the crystal structure:
| Substitution | Prediction | Database |
|---|---|---|
| Gln172His | neutral | dbSNP |
| Ala259Val | non-neutral | HGMD |
| Thr266Ala | non-neutral | dbSNP |
| Phe392Ser | non-neutral | dbSNP |
| Pro416Gln | non-neutral | HGMD |
Gly103Ser
...
Gln172His
...
Ala259Val
...
Thr266Ala
...
Phe392Ser
...
Pro416Gln
...
Mutated structure creation
SCWRL
...
FoldX
...
Energy comparisons
...
Minimise
In the table below, the energy for all five runs of the minisation are given. Since the SCWRL output could not be minimised, we only can see the difference between the wildtype (WT) and the five mutation structures constructed with foldX. <figtable id="pro-struc">
| minimisation run | |||||
|---|---|---|---|---|---|
| Type | 1 | 2 | 3 | 4 | 5 |
| WT | -7516.27 | -7524.20 | -7291.36 | -7133.71 | -6996.34 |
| Q172H | -7514.27 | -7504.92 | -7281.60 | -7131.31 | -7023.56 |
| A259V | -7469.61 | -7462.48 | -7221.58 | -7065.94 | -6951.32 |
| T266A | -7536.77 | -7523.38 | -7298.14 | -7165.29 | -7084.60 |
| F392S | -7511.51 | -7528.61 | -7290.01 | -7132.75 | -7010.52 |
| P416Q | -7556.57 | -7542.79 | -7299.39 | -7151.21 | -7040.37 |
</figtable> The energies of the wildtype and the mutated structures is very similar and is per run increasing slightly. Only for the structures of the wildtype and the mutation F392S has the second run a small decreased value.
References
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