I would like to see this puzzle in different sizes, like 25, 35, and 45 residues with the 3 cystine bond pattern requirement! I think that would give you guys a nice big set of good designs to pick from.

There's more info and diagrams for some "leapfrog" disulfide patterns here: http://www.cyclotide.com/knots.html

The page says cystine knots are 26-48 residues long, so 25 may be at the lower limit of what will fold up that way. I agree with wisky - I'd like to work on longer versions too.

Also would like to be able to remove and insert residues from this protein

me also, inserting gives some creative options, as it is this one is pretty close to what was given.

I know that 25 residues is definitely a challenge. We picked this size for two reasons: first, it's in a size range that is nicely compatible with chemical synthesis. Large proteins must be expressed in bacteria or yeast, but with a small enough peptide, we have the option of artificially synthesizing the peptide. This allows for inclusion of nonstandard amino acids and other unnatural modifications that can add new functionality, as well as for large-scale production of fairly pure peptide. Unfortunately, the longer the peptide, the harder it is to synthesize and get good yields and high purity. I wanted to keep the peptide short enough that synthesis is a very feasible option.

Second, where large proteins are difficult to get into the body and difficult to get across barriers, a sufficiently small peptide (particularly if compact and ordered) has the potential for oral bioavailability, which makes it easier to use as a drug.

In short, if we keep it small, we might have some of the advantages of both small-molecule drugs (which get into the body easily and are easy to produce in large amounts, but which are usually pretty nonspecific) and proteins (which can have terrific specificity and affinity for a target, meaning a potent desired effect with few side-effects, but which don't get into the body easily). That's the hope, anyways!

We have advanced the deadline for this puzzle by several hours to accommodate a scheduled server outage: http://fold.it/portal/node/998369