Update on Aflatoxin Challenge

The Siegel Lab is back again with an update on the Foldit Aflatoxin Challenge!

You may remember back in September of last year we came to you with Puzzle 1739: Aflatoxin Challenge: Round 13, based on a new enzyme scaffold that was shown to be active on aflatoxin.

We were really thankful for all of your designs and even gave a few back to you as prediction-style puzzles in Rounds 14-17. These puzzles challenged you to predict the apo structure of the designs -- the protein structure when aflatoxin is not present. We have some news regarding our results, now that we have transferred them from in silico to in vitro!

Prediction of apo structures

When we compiled all of your puzzle solutions from Round 13 and narrowed it down to the most promising entries, we wanted to do a pilot study of a few of the most radical designs to determine how well the laccase starting protein would handle large, structural changes.

Many of these designs expressed and were active in our reporter assay, which we were happy to see; however, all had lost the ability to degrade aflatoxin. We believed that the active site was changed in a way that didn’t allow aflatoxin to fit for catalysis. Your solutions for our apo structure puzzles readily confirmed this.

Below, the original player design is shown in green, with the other colors depicting the best scoring player apo solutions. We can see that these top-scoring apo structures are crowding the position where aflatoxin is supposed to sit. Clearly aflatoxin would have a hard time fitting in those active sites, which was very consistent with what we were seeing in our assays.

Two problematic designs from previous experiments. In the absence of aflatoxin, these designs are likely to fold into alternate structures that would have difficulty binding the aflatoxin molecule.

New testing results

Using this information we ordered 56 player designs from Puzzle 1739 and tested them all using our high throughput methods. Fortunately, several had activity on aflatoxin and all of these were grown and assayed in larger scale to ensure accuracy.

Of the approximately 20 active designs, 3 were found to be the most active and highest expressing, making them excellent candidates for more design! Two of these come from LociOiling, and the third is a design by Phyx.

We want to understand how the active sites for these 3 designs may look when aflatoxin is not present, so we are releasing new apo prediction puzzles based on these designs in the coming weeks. We hope you will give these puzzles a try and help us in the next step of the Aflatoxin Degradation Project!

Play Puzzle 1902: Aflatoxin Challenge: Round 18 Loop Prediction now!

( Posted by  bkoep 85 703  |  Tue, 10/06/2020 - 21:52  |  0 comments )
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