Journal club: Principles for designing ideal protein structures

The goal of protein design is to generate a sequence that assumes  a certain structure and/or performs a specific function. A recent paper in Nature has attempted to design sequences for each of five naturally occurring protein folds. The success rate ranges from 10-40%.

This recent work comes from the Baker group, who are best known for Rosetta and have made several previous steps in this direction. In a 2003 paper this group stripped several naturally occurring proteins down to the backbone, and then generated sequences whose side-chains were consistent with these backbone structures. The sequences were expressed and found to fold into proteins, but the structure of these proteins remained undetermined. Later that same year the group designed a protein, Top7, with a novel fold and confirmed that its structure closely matched that of the design (RMSD of 1.2A).

The proteins designed in these three pieces of work (the current paper and the two papers from 2003) all tend to be more stable than naturally occurring proteins. This increased stability may explain why, as with the earlier Top7, the final structures in the current work closely match the design (RMSD 1 or 2A), despite ab initio structure prediction rarely being this accurate. These structures are designed to sit in a deep potential well in the Rosetta energy function, whereas natural proteins presumably have more complicated energy landscapes that allow for conformational changes and easy degradation. Designing a protein with two or more conformations is a challenge for the future.

In the current work, several sequences were designed for each of the fold types. These sequences have substantial sequence similarity to each other, but do not match existing protein families. The five folds all belong to the alpha + beta or alpha/beta SCOP classes. This is a pragmatic choice: all-alpha proteins often fold into undesired alternative topologies, and all-beta proteins are prone to aggregation. By contrast, rules such as the right-handedness of beta-alpha-beta turns have been known since the 1970s, and can be used to help design a fold.

The authors describe several other rules that influence the packing of beta-alpha-beta, beta-beta-alpha and alpha-beta-beta structural elements. These relate the lengths of the elements and their connective loops with the handedness of the resulting subunit. The rules and their derivations are impressive, but it is not clear to what extent they are applied in the design of the 5 folds. The designed folds contain 13 beta-alpha-beta subunits, but only 2 alpha-beta-beta subunits, and 1 beta-beta-alpha subunit.

An impressive feature of the current work is the use of the Rosetta@home project to select sequences with funnelled energy landscapes, which are less likely to misfold. Each candidate sequence was folded >200000 times from an extended chain. Only ~10% of sequences had a funnelled landscape. It would have been interesting to validate whether the rejected sequences really were less likely to adopt the desired fold — especially given that this selection procedure requires vast computational resources.

The design of these five novel proteins is a great achievement, but even greater challenges remain. The present designs are facilitated by the use of short loops in regions connecting secondary structure elements. Functional proteins will probably require longer loops, more marginal stabilities, and a greater variety of secondary structure subunits.

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