Tag Archives: protein design

3 Key Questions to Think About When Designing Proteins Computationally

We have reached the era of design, not just ‘hunting’. Particularly exciting to me is the de novo design of proteins, which have a wide and ever increasing range of applications from therapeutics to consumer products, biomanufacturing to biomaterials. Protein design has been a) enabled by decades of research that contributed to our understanding of protein sequence, structure & function and b) accelerated by computational advances – capturing the information we have learned from proteins and representing it for computers and machine learning algorithms.

In this blog post, I will discuss three key methodological considerations for computational protein design:

  1. Sequence- vs structure-based design
  2. ML- vs physics-based design
  3. Target-agnostic vs target-aware design
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Highlights from the European Antibody Congress 2021

Last month, I was fortunate enough to be able to attend (in person!) and present at the Festival of Biologics European Antibody Congress (9-11 November, 2021) in Basel, Switzerland. The Festival of Biologics is an annual conference, which brings together researchers from industry and academia. It was an excellent opportunity to learn about exciting research and meet people working in the antibody development field.

Here are some of my highlights from the European Antibody Congress, with a focus on antibody design and engineering:

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Miniproteins – small but mighty!

Proteins come in all shapes and sizes, ranging from thousands of amino acids in length to less than 20. However, smaller size does not correlate with reduced importance. Miniproteins, which are commonly defined as being less than 100 amino acids long, are receiving increased attention for their potential roles as pharmaceuticals. A recent paper by David Baker’s group put miniproteins into the spotlight, as the study authors were able to design miniproteins that bind the SARS-CoV-2 spike protein with as strong affinity as an antibody would – but in a tiny fraction of the size (Cao et al., 2020). These miniproteins are much cheaper to manufacture than antibodies (as they can be expressed in bacteria) and can be highly stable (with melting temperatures of >90º possible, meaning they can easily be stored at room temperature). The most promising miniprotein developed by the Baker group (LCB1) is currently undergoing testing to be used as a prophylactic nasal spray that provides protection against SARS-CoV-2 infection. These promising results – and the speed in which progress was made – brings the vast potential of miniproteins in healthcare to the fore.

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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.