The first round of results on designed fibronectin are in....
We have previously described the design problem of improving fibronectin and we chose to produce a design by BootsMcGraw. The results are in, but unfortunately the product did not show an improvement in stability. The bacteria made the protein just fine, but it was largely insoluble. This happens usually when the protein produced by the bacteria is not properly folded and started to aggregate as it was being produced so they never had the chance of folding to the target structure. What usually happens next is that a biochemist will carefully solubilize the protein and refold it by keeping it at a low concentration while reducing the amount of denaturants. This way the protein will hopefully fold to its shape first before clumping together and crash out of the solution. To be thorough, our collaborator at USC did exactly that, but we never obtained any soluble protein even after this process.
As described previously, the design by BootsMcGraw was chosen because it has the most balanced qualities according to our force field, namely it was very well packed, got good scores, and was evaluated to have few unsatisfied hydrogen bonding groups. We knew from the start that the sequence was more hydrophobic than what one would expect from an all-beta protein, but we decided to try it since the model buried the hydrophobic groups quite nicely and it was folded correctly by the automated fold-prediction algorithm. The experimental evidence suggests that the scores can be deceptive and if we allow backbones to move relatively freely, it was easy for users to over-pack the core and made them insoluble. This is of course only part of the problem, and the same happens when we run automated design. There were also published evidence that the loops we allow users to mutate may be important for stability (we allow users to change them since FoldIt is setup for users to explore the folding landscape, we are hoping to get better answers than the wildtype). All these should be taken into account for our next round.
Subsequently we've posted a puzzle on the same protein. we locked the backbone structure while imposing constraints to allow mutations only within the amino acids used by proteins with high homology. The results we recovered were quite encouraging, since we got back designed sequences very much like the wildtype even when a poly-alanine was given to the users to start. Interestingly, after collecting the top designed sequences, we realized that most of the mutations suggested by FoldIt players were already tested in a research paper. Some mutations maintained the stability, but none of which improved it.
We are tweaking the puzzle and will try again soon.
happy holidays.
Possu
( Posted by
possu 127 6665 |
Tue, 12/22/2009 - 19:30 |
4 comments
)
Demographic Survey
We are interested in learning more about who plays Foldit. If you'd like to help us out, you may complete this very short demographic survey:
http://fold.it/portal/node/987033
Thank you for your time!
( Posted by Seth Cooper 127 4110 |
Fri, 11/27/2009 - 19:34 |
0 comments
)
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 127 6665 |
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.
credits:
Design: BootsMcGraw, Texas.
BOINC: Firas Khatib, Baker Lab, UW.
Experiment: Terry Takahashi, Roberts Lab, USC.
possu 127 6665 |
Fri, 10/16/2009 - 21:49 |
7 comments
)
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.
