Canavan Disease: Task 09 - Structure-based Mutation Analysis

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Revision as of 14:36, 1 September 2013 by Boehma (talk | contribs) (Comparisons)

Structure-based mutation analysis looks at the effects of mutations on the protein structure. This is done via the calculation of energetic changes.

LabJournal

Preparation

Several pdb-structures are available for aspartoacylase. To work on structure based muatation analysis, the one to choose should have some classifications: The structure should have a high resolution with a small R-factor. The pH value should be near the physiological pH and there should be no missing gaps.
There was no structure without any missing gaps, but the missing gaps were only at the beginning and end of the structure. Only one pdb-structure contained further information such as pH value. Therefore 2O4H was chosen: It is the human brain aspartoacylase complex with intermediate analog (N- 2 phosphonomethyl-L-aspartate), with a resolution range of 2.7 Angstroms. It is one single christal analyzed with X-ray diffraction in October 2006 using a temperature of 100 Kelvin and a pH of 6.0. Its free R value is 0.271. The missing residues range from positions 1-9 in the beginning and 311-313 in the end of the protein.

Visualization

Some mutations from the previous task were chosen to work with. Those are displayed in <xr id="visu"></xr>:

<figtable id="visu">

Visualization of Mutations of Interest
Mutation Description Visualization Sec. Struc. Information
Asn121Asp From the previous task it is known that Asn->Ile is disease causing. Here a detailed look on that specific mutation is of interest.
CanvanT9 Mutation Asn121Asp.png
LOOP possibly disease causing
His21Pro This mutation is definitively disease causing, since it is important for binding the zinc atom. The position is part of the active center and therefore of special interest.
CanvanT9 Mutation His21Pro.png
LOOP metal binding,
disease causing
Pro149Ala Since Proline is known to be a HELIX breaker and this mutation is very near to a HELIX the Proline might be necessary for the structure.
CanvanT9 Mutation Pro149Ala.png
LOOP
near HELIX
-
Pro257Arg According to Task 8 the mutation is expected to have no effect on the protein.
CanvanT9 Mutation Pro257Arg.png
LOOP -
Thr166Ile This position is known to be part of the binding region. It is listed to be disease causing and is exactly the changing point between LOOP and HELIX.
CanvanT9 Mutation Thr166Ile.png
LOOP
near HELIX
in binding region,
disease causing
Visualization of Mutations in 2O4H(orange). The mutated sequence is colored blue.
Information about functional residues were taken from Uniprot.

</figtable>

Comparisons

Comparing wildtype to mutant structures no potential clashes were found in neither of the mutations. No holes were introduced to the structure. The mutations should not have a high effect on changing hydrophilicity in the core, nor hydrophobicity in the surface. Only Thr->Ile, which is part of the core region changes extreme from a more hydrophilic to a more hydrophobic aminoacid. A visual comparison can be found in <xr id="engy"></xr>.

<figtable id="engy">

Energy Comparisons
Mutation Description H-bonds SCWRL and FOLDX
Asn121Asp Mutating the sidechain leads in both approaches (mutate via Pymol and create a new structure with SCWRL or FOLDX) to a loss of one potential hydrogen bond. However, as the residue is not located within a secondary structure element that relies on stabilization via H-bonds, the impact is not expected to be extreme.
Canavan Mutation Asn121Asp hbond.png
Canavan Scwrl FoldX Asn121Asp.png
His21Pro description
Canavan Mutation His21Pro hbond.png
CanavanScwrl-foldx His21Pro.png
Pro149Ala description
Canavan Mutation Pro149Ala hbond.png
Canavan Scwrl FoldX Pro149Ala.png
Pro257Arg description
Canavan Mutation Pro257Arg hbond.png
Canavan Scwrl FoldX Pro257Arg.png
Thr166Ile description
Canavan Mutation Thr166Ile hbond.png
Canavan Scwrl FoldX Thr166Ile.png
For the visualization of HBonds, the original sequence is colored green and the mutated sequence blue. Comparing SCRWL (red) and FOLX (cyan) is another aaporach

</figtable>

Energy Comparison

<figtable id="energy_comp">

Energy comparison
Mutation SCWRL foldX
MT Corr. MT Corr.
Wildtype 400.00 144.46
Asn121Asp 403.94 1.01 145.46 1.01
His21Pro 395.75 0.99 145.85 1.01
Pro129Ala 401.93 1.00 148.04 1.02
Pro257Arg 414.53 1.04 149.63 1.04
Thr166Ile 142.25 1.08 147.35 1.02
Energy Comparison

</figtable>

Minimization

Canavan Minimization.png

Supplement

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