Week 6 Assignment

Today I’ll be looking at the 3D structure of my pet gene ASPA. When looking through the NCBI structure database I decided on using the entry with the PDB ID of 2O53. It was one of the most recent 3D models and appeared to have to no alterations. This protein came as a dimer but I hid one of the regions to better examine my target area. On my last post I mentioned the E285A mutation that has been found to cause Canavan disease and I compared the wild type protein to the E285A variant with various structural modeling algorithms. Today I’ll be looking at the 285th AA in the actual 3D model.

Below you can see the space filling model. While hard to see the highlighted region is in a small concave on the 3D model. This is believed to be where the catalytic domain is.

In this next picture is a little bit closer examination of the target spot. From here you can see that the 285 spot is part of a turn between two beta sheets.

When actually examining the 3D structure I found quite a few of the predictions the algorithms gave me in my last post were incorrect or slightly off. First off the 3D models shows that the 285 region is actually a simple turn as you can see above. The Protean 3D algorithm guessed that area would be an alpha or beta region and not a turn. Protean 3D also predicted that the 285 area would be hydrophobic and the area directly after would be hydrophilic. As you can see from the picture below the 285 region is actually hydrophilic. The predictions weren’t totally wrong because the model does show a hydrophobic region followed by a hydrophilic region just a few AAs down the protein.

The charge region was also a little off as you can see in the picture below. The 285 spot is negative as predicted but Protean 3D also predicted that a few AAs on each side would be negative. But in the 3D model it shows that the AAs around the 285 spot are neutral.

Overall I would say that the Protean 3D algorithms had some issues predicting what this protein would actually look like. The predictions were definitely helpful when seeing what the E285A variant would do but the actual 3D model has quite a few differences.

Week 5 Assignment

The first allelic variant from for aspartoacylase (ASPA) in omim is an 854 A-C transversion. A transversion is the substitution of a purine for a pyrimidine. This causes a glutamic acid to alanine switch at the 285 spot. Alanine is one of the simplest AAs with a functional group composed of only a methyl. The methyl on alanine is non reactive so you will almost never see alanine directly involved in protein function. Glutamic Acid on the other hand is highly reactive and found in many protein functions. So what is happening is the replacement of a highly reactive AA with an essentially non reactive AA.

The substitution at 285 is predicted to be part of the catalytic domain of ASPA. This mutations was found in 18 Ashkenazi Jew patients with Canavan disease. Of 879 Ashkenazi Jew scanned in the Elpeleg et all (1994) study 1 in 59 were found to be heterozygote carriers. This study showes the importance for Ashkenazi Jews to scan for this mutation.

Below is the reaction that ASPA takes part in which will play an important role when discovering how the structural changes, of the E to A substitution, affects this enzyme.

ASPA is a hydrolase enzyme whose reaction looks like this: N-acyl-L-aspartate + H₂O a carboxylate + L-aspartate

Using Protean 3D the first thing you will notice is that according to the Chou-Fasman secondary structure analysis the E285A variant appears to have a higher likely hood of a beta region at 285. Which when it comes to protein interactions the shape of the protein is very important. So having structural changes at what is believe to be the catalytic domain will mostly likely result in less or different binding.

Another change and what I believe to be the most significant is the change to the hydrophobicity. According to both Kyte-Doolittle and Hopp-Woods the E285 variant becomes more hydrophobic. So with ASPA being a hydrolase enzyme and relying on water for its catalytic affect making the catalytic domain more hydrophibic is going to play a substantial  role in how well ASPA is able to catalyze its reaction. Protean also shows the ASPA E285A variant to be more hydrophobic. Also according to Yasanandana S. Wijayasinghe et all

“The structure of the E285A mutant, the most common clinical mutant, reveals that the loss of hydrogen bonding interactions with the carboxylate side chain of Glu285 disturbs the active site architecture, leading to altered substrate binding and lower catalytic activity.”

The last difference between ASPA and ASPA E285A according to Lehninger charge density plot is the negative region. Which is obviously due in part to the difference between Glu and Ala. Glu has a carboxylic acid functional group while Ala has a methyl group. Obviously the carboxylic acid group is going to be much more negatively charged. This is going to play into the hyphobicity difference we saw above.

A few things didn’t change with the substitution. The antigenicity according to the Jameson-Wolf plot did not change. This makes sense because the structural change was to such a small area of the protein. Also the flexibility remained the same. This I believe is another case of the change being so small but to such an important area.

Over all the E285A variant is such a small change when not looking at the 3D structure. It is only a single AA change in a protein that is 314 AAs long. But that change unluckily falls on the catalytic domain and turns ASPA into a much more hydrophobic enzyme unable to catalyze to its full potential.