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.