NextGen protein design

Abstract

Protein engineering is at an exciting stage because designed protein-protein interactions are being used in many applications. For instance, three designed proteins are now in clinical trials. Although there have been many successes over the last decade, protein engineering still faces numerous challenges. Often, designs do not work as anticipated and they still require substantial redesign. The present review focuses on the successes, the challenges and the limitations of rational protein design today.

Figure 1. Redesign of globular proteins

In (A)–(C), the top image shows a first-generation protein and the bottom image shows a second-generation protein. First-generation proteins are shown in (D) and (E). A cartoon representation of each protein is shown in grey. Positions randomized in first-generation libraries are shown as red spheres, new randomized positions in second-generation libraries are shown as orange spheres, positions mutated for other reasons (e.g. to improve stability or hydrophilicity) are shown as blue spheres, and large protein segments fused to first-generation scaffolds are shown as green cartoons. (A) Monobody (PDB code 1TTG): second-generation monobodies include mutations that change the location of the randomized binding site. (B) Affibody (PDB code 3MZW): second-generation Affibodies include mutations to increase biophysical properties and reduce cross-reactivity with the native ligand IgG. (C) PDZ domain [PDB codes 1BE9 (top) and 3CH8 (bottom)]: second-generation PDZ designs include the fusion of a monobody to a circularly permuted PDZ domain. Peptide ligands are shown as light blue sticks. (D) SH3 domain (PDB code 5HCK). (E) WW domain (PDB code 2LB3)

Figure 2. Redesign of repeat proteins

In each panel, the top image shows a first-generation protein and the bottom image shows a second-generation protein. A cartoon representation of each protein is shown in grey. Positions randomized in first-generation libraries are shown as red spheres, new randomized positions in second-generation libraries are shown as orange spheres, positions mutated for other reasons (e.g. to improve stability) are shown as blue spheres and large protein segments fused to first-generation scaffolds are shown as green cartoons. (A) TPRs (PDB code 3KD7): second-generation consensus TPRs include mutations that optimize the charged character of the convex (non-binding) face of the TPR. (B) LRRs (PDB code 2Z65): second-generation LRRs include two point mutations that improve ligand-binding affinity 3000-fold. (C) DARPins (PDB codes 1MJ0 and 2XEE): second-generation DARPins may incorporate a stabilized C-terminal cap into first-generation libraries.