Abstract

Protein structure design and engineering is a research endeavour in which proteins with predicted structure and function are
constructed in the laboratory through rational design, combinatorial selection or combination of both approaches. It is built
upon our knowledge about the structure and function of proteins and can be accomplished either from scratch (de novo design) or based on native scaffolds (redesign). The area of protein design is an exciting and rapidly growing field, advancing
from the design of simple protein structures, to those that are more complicated and recently to the designs of functional
proteins. Design of artificial proteins containing unnatural amino acids, backbone linkages or cofactors have also been reported,
making it possible to prepare proteins with structural and functional properties beyond those of native proteins. These advances
bring us closer to realising the dream of tailor‐made artificial enzymes with high catalytic efficiency and selectivity for
biotechnological and pharmaceutical applications.

Key Concepts:

Protein structure design and engineering is a research endeavour in which proteins with predicted structure and function are
constructed in the laboratory.

The protein design field can be organised into two complimentary approaches: rational design and combinatorial selection of
the desired protein.

Rational protein design strategies can involve designing a protein from scratch (de novo design) or redesigning a protein with native scaffolds to achieve new structures and functions.

Combinatorial approaches can involve sampling a large population of proteins to select the desired one or it can involve numerous
rounds of randomised mutation followed by selection to fine‐tune properties being selected for.

Techniques are available that allow the incorporation of unnatural moieties, such as unnatural amino acids, backbone linkages
or cofactors, into proteins.

The field has made a big stride recently in designing functional proteins.

Stereoview of the X‐ray crystal structure (red) of the unique de novo designed 93‐residue α/β protein called Top7, which was determined to be very similar to the computationally designed model
(blue) determined by the Rosetta program. Reproduced with permission from Kuhlman et al..

Figure 2.

The rational redesign of myoglobin to a nitric oxide reductase: overlay of the 1.72Å resolution crystal structure (blue) of a designed FeB‐Mb centre and the computer model (yellow). The designed protein was found to mimic the structure and function of native nitric
oxide reductase.
Reproduced with permission from Yeung et al..

Figure 3.

The first protein crystal structure derived from function‐directed in vitro evolution. It has a Zn(ii)‐binding as well as an ADP‐binding site. PDB ID=1UW1. Reproduced with permission from Lo Surdo et al..

Figure 4.

The semisynthetic technique of expressed protein ligation was used to vary the active site Met121 and Cys112 residues in the blue copper protein azurin with iso‐structural amino acids to probe the electronic contributions of the ligands
without altering steric parameters. Reproduced with permission from Garner et al..

Figure 5.

Overlay of crystal structure (cyan) and computationally designed structure (grey) of the active site for a prototypical Kemp
elimination catalyst (removes an hydrogen ion from carbon). The final protein was found to accelerate the reaction rate by
105. Further rounds of directed evolution increased the rate parameters by 200 times, nicely demonstrating the combined use of
de novo design followed by combinatorial directed evolution techniques. Reproduced with permission from Röthlisberger et al..