Towards functional de novo designed proteins

Our ability to design completely de novo proteins is improving rapidly. This is true of all three main approaches to de novo protein design, which we define as: minimal, rational and computational design. Together, these have delivered a variety of protein scaffolds characterised to high resolution. This is truly impressive and a major advance from where the field was a decade or so ago. That all said, significant challenges in the field remain. Chief amongst these is the need to deliver functional de novo proteins. Such designs might include selective and/or tight binding of specified small molecules, or the catalysis of entirely new chemical transformations. We argue that, whilst progress is being made, solving such problems will require more than simply adding functional side chains to extant de novo structures. New approaches will be needed to target and build structure, stability and function simultaneously. Moreover, if we are to match the exquisite control and subtlety of natural proteins, design methods will have to incorporate multi-state modelling and dynamics. This will require more than black-box methodology, specifically increased understanding of protein conformational changes and dynamics will be needed.

Introduction

De novo protein design is said to have come of age [1]. From the early de novo proteins confirmed by high-resolution structures [2, 3, 4], the field has advanced rapidly with new scaffolds covering all-α [5,6^•,7], all-β [8], and mixed-α/β and α + β structural space [9,10^•,11]. In addition, side-chain constellations can be controlled exquisitely to introduce networks of hydrogen bonds throughout target structures [12], which, in turn, can improve the design and characterisation of de novo membrane proteins [13].

However, the ability to design functional de novo proteins from scratch, or to embellish existing de novo scaffolds with new functions, is still in its infancy. Herein, we use terms like ‘functional protein design’ for any stably folded de novo protein frameworks that incorporate interactions with small or large molecules, catalytic activity and so on. With notable exceptions—for example, reports of a functional ion transporter [14], a de novo designed catalytic triad [15^••], and a highly efficient de novo enzyme [16^••]— general design principles for functional protein design are sparse. Indeed, it may be that overoptimised de novo proteins, which are often hyperthermally stable, may not make good platforms for functional design, as it is known that dynamics play essential roles in ligand binding and catalysis [17, 18, 19].

Herein, we focus on truly de novo proteins rather than those achieved through protein engineering or redesign—that is, where functions are improved in or introduced to natural proteins. Of course, the latter have led to novel enzymes and ligand-binding proteins [20, 21, 22]. Whilst impressive, protein engineering relies on the inherent stability of natural scaffolds and their tolerance to mutation, and often uses the randomness of directed evolution to access the targeted function [23]. By contrast, de novo protein design removes the dependence on naturally evolved scaffolds, and has the potential for a deeper understanding of the contribution that every side chain makes towards the structure, stability and function of de novo proteins. Of course, this is an extremely challenging approach and its goals are ambitious.

Notable advances have also been made in introducing metal-binding and protein–protein interactions into de novo proteins (see recent reviews Refs. [24, 25, 26]). However, these pose different challenges to those laid out herein, and are only mentioned in passing in this review.