Pack the Holes and Fight Cancer!
As the quest to develop new ways of fighting cancer continues, methods such as prodrug therapy are promising but currently lack the ideal tools necessary for effective treatment. One such tool is a non-immunogenic enzyme that catalyzes the deamination of cytosine, converting it to uracil. There is clinical need for a human cytosine deaminase for use as a prodrug activator in suicide gene therapy, where a cytosine deaminase can catalyze the reaction of the cytosine derivative 5-fluorocytosine (a harmless chemical) into 5-fluorouracil (a cytotoxin). While there are cytosine deaminases that exist in nature, there is no human enzyme capable of catalyzing this reaction. The use of non-human enzymes in prodrug therapy often fails due to the immunogenicity issues that can arise. Therefore, by taking a human enzyme and altering its specificity such that it can catalyze the deamination of cytosine, these immunogenicity issues can be avoided.
The “Pack the Holes and Fight Cancer” puzzles are based on a previously designed human guanine deaminase in which its specificity was successfully changed from guanine to ammelide (which has a structure is very similar cytosine) by modification of a catalytic loop. This original re-design left a few large holes near the loop and ligand. Thus, to increase the activity of the enzyme and move closer to the final goal of creating a human cytosine deaminase, we need to pack these holes! There are two puzzles that offer different packing possibilities.
Your designs will be ranked based on score, packing, and number of additional contacts made to stabilize the loop residues 119-121 (particularly the sidechain of N120). The most promising of your designs will then be synthesized and experimentally tested in the Baker lab. We look forward to seeing all of the exciting new solutions you come up with!( Posted by JMDNivala 82 2383 | Tue, 11/10/2009 - 00:33 | 0 comments )
Will this work? We'll see....
One of the most recent design puzzles posted is a human fibronectin
structure (from PDB: 1FNF). It is an all-beta protein that is commonly used
as a scaffold for making antibody mimetics. The reason for using
fibronectin is that it does not contain the disulfides commonly found in
antibodies, and is stable in reducing conditions. Most importantly they can
be produced in large quantities in E. coli. A collaborator of ours takes
advantage of these properties, using fibronectin to host a variety of loops
on its surface and panning for desirable properties in a selection
experiment. It has been observed, however, that the stability of
fibronectin sometimes is drastically reduced when their loops connecting the
strands are replaced with non-native ones, as in engineering fibronectin to bind a particular target.
A considerable amount of protein (and therefore the diversity one can screen)
can be lost due to this. An ideal scenario would be to have an extremely
stable scaffold for which the stability is independent of the binding loops,
therefore the experiments are really testing the properties of the loops.
Just to draw an analogy, imagine you are testing the sharpness of knifes,
but the handles on them randomly fail so when you cannot drive a particular
blade through an object, you never know whether it was sufficiently sharp.
We put out this fibronectin puzzle to see if players can pack the core more
tightly, and in the end we chose a single structure produced by the FoldIt
community for experimental testing. It was the design done by BootsMcGraw.
It was chosen because the model showed very promising traits, with few
holes in the core and is scored relatively well. The top ranking structures
sometimes have an excessively abundant aromatic residues, so we did not try
those. To validate the design before it goes into production (which is
time-consuming, and costly, by the way), we sent the sequence through our
structure prediction algorithm via BOINC, and interestingly it found a
funnel near the native structure (the starting structure in this case).
When compared to the funnel generated with the native sequence found on the
crystal structure, they are quite similar! (see figure) Our collaborator at USC has
agreed to test this out in the lab. We'll soon know if this first-ever
experimentally tested design from FoldIT has an improved stability.
Design: BootsMcGraw, Texas.
BOINC: Firas Khatib, Baker Lab, UW.
Experiment: Terry Takahashi, Roberts Lab, USC.
new puzzle: finding home
The new puzzle "finding home" features a homing endonuclease recognizing its target DNA sequence. Homing endonucleases are proteins involved in transfer and replication of DNA sequences, in most of the cases their own. They recognize specific sites in the genome and are able to cut them precisely at that point and trigger the insertion of a new copy of their own gene. The surface in contact with the DNA is responsible for the recognition and discrimination of DNA sequences. Currently there is a lot of interest in homing endonucleases as tools for gene therapy, replacing mutated genes involved in diseases with correct copies. The challenge is to change the specificity of homing endonucleases to recognize new DNA sequences.
And this could be the topic of some of the future puzzles.
Selection Mode Feedback
A couple months back, we released an update that allowed you to try out a new interface that we have been working on that was based upon selecting portions of the protein and perform actions on that selection. (Review of that update can be found at http://fold.it/portal/node/986573). We're interested to hear what those who have tried out this new interface think about it, and whether or not it is or could be an improvement over the existing interface.
Please comment with your feedback, or simply vote on this post based on whether you like the new interface or not.( Posted by beenen34 82 2383 | Thu, 10/08/2009 - 22:58 | 4 comments )