Novel proteins: from fold to function

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The field of de novo protein design, though only two decades old, has already reached the point where designing and selecting novel proteins that are functionally active has been achieved several times. Here we review recently reported de novo functional proteins that were developed using various approaches, including rational design, computational optimization, and selection from combinatorial libraries. The functions displayed by these proteins range from metal binding to enzymatic catalysis. Some were designed for specific applications in engineering and medicine, and others provide life-sustaining functions in vivo.

Highlights

► We review recent advances on de novo proteins that are functionally active. ► Approaches for developing functional de novo proteins include rational design, computational optimization, and selection from combinatorial libraries. ► Functions range from metal-binding and catalysis to life-sustaining functions in vivo. ► Potential applications include nanotechnology, engineering, and medicine.

Introduction

The sequences and structures of natural proteins are the results of eons of evolutionary selection. Some features of these proteins are crucial for their functions, while others are merely ‘evolutionary baggage’ that came along for the ride. Designing proteins de novo provides an opportunity to separate the crucial from the coincidental. Design also allows scientists and engineers to explore beyond what has already appeared in nature, and to devise structures and functions that are possible, but have not yet been sampled by nature. In just over 20 years, since the first de novo designed proteins were reported [1, 2], many different structures have been described [3]. Some are recapitulations of three-dimensional structures that occur frequently in nature, while others were designed to fold into topologies that had not been seen previously [4, 5, 6]. Although the design and optimization of stable structures continues as an active research area [7], the next step  incorporating functional activity into de novo proteins  is becoming a major focus of the field.

This review will focus on proteins that are not based on natural sequences. We emphasize recent achievements; readers are advised to consult other reviews for discussions of earlier work on the binding activities of de novo proteins and peptides [6, 8, 9].

Section snippets

Proteins designed to bind metals

One of the simplest protein functions is binding, and the simplest ligand bound by native proteins is a metal ion. Indeed, nearly a third of natural proteins contain a metal-binding site [8]. Thus, it is not surprising that some of the first functional de novo proteins were designed to bind metals such as zinc or mercury [10, 11]. One class of these metal-binding proteins was based on a helix-loop-helix dimer and known as the duo-ferri (DF) proteins because the earliest versions bound two irons

Proteins designed to bind targets ranging from small cofactors to large receptors

Four-helix bundles are relatively easy to design, and numerous functions have been designed onto this structural scaffold. In most cases, the structure was designed first, and function was added in a subsequent stage. A function that has been explored extensively in four-helix bundles is the ability to bind heme and related porphyrins [19, 20, 21]. One de novo four-helix bundle protein was altered to bind heme simply by adding four histidine residues at appropriate positions [22]. A variant was

Beyond binding: novel proteins for catalytic and biological functions

Proteins can be designed to mimic functions that occur in very specific tertiary structures. For example, a de novo protein designed to mimic the rubredoxin β-sheet structure was shown to bind iron and remain stable for 16 cycles of oxidation–reduction [29]. In another example, a library of proteins was designed to fold into the secondary and tertiary structure of the helical bundle protein chorismate mutase, using a limited library of possible amino acids. Using a selection in chorismate

Functional proteins from combinatorial libraries of novel sequences

An alternative approach to residue-by-residue rational design is to construct large libraries of novel sequences and then screen for function. If the libraries are constructed randomly, then the vast majority of sequences will not be functional, and finding rare functional sequences will require screening through enormous libraries. Nonetheless, a pioneering study by Keefe and Szostak selected four ATP-binding proteins from a random library containing 6 × 1012 sequences 80 amino acids in length [

Novel proteins that function in vivo

Although the field of protein design has focused primarily on devising novel proteins that function in vitro, a long-term goal is to produce novel macromolecules that provide essential cellular functions in living systems. A major advantage of working with activity in vivo is that one does not have to rely on engineered screens. Instead, one can use more powerful life-or-death genetic selections. Our laboratory has used selections in vivo to probe a library of 1.5 × 106 novel four-helix bundles

Conclusion

De novo proteins offer promise in many areas of research, from basic biology to applications in engineering and medicine. Design can be used to increase activity, enhance protein stability and shelf life, decrease protein size, and uncover information about the mechanisms of reactions. Moreover, compared to standard organic chemistry procedures, protein catalysts are environmentally more benign. Increased computational power and better modeling allow more of the work to be done rapidly before

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgement

This work was funded by NSF grant MCB-0817651.

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