Novel proteins: from fold to function
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.
References (51)
- et al.
Molecular recognition with designed peptides and proteins
Curr Opin Chem Biol
(2005) - et al.
Probing metal–protein interactions using a de novo design approach
Curr Opin Chem Biol
(2005) - et al.
Mimicking photosynthesis in a computationally designed synthetic metalloprotein
J Am Chem Soc
(2003) - et al.
Redox characteristics of a de novo quinone protein
J Phys Chem B
(2007) - et al.
An artificially designed pore-forming protein with anti-tumor effects
J Biol Chem
(2003) - et al.
De novo computational design of retro-aldol enzymes
Science
(2008) - et al.
Physical organic-chemistry of benzisoxazoles. 1. Mechanism of base-catalyzed decomposition of benzisoxazoles
J Org Chem
(1973) - et al.
De Novo designed proteins from a library of artificial sequences function in Escherichia coli and enable cell growth
PLoS ONE
(2010) - et al.
Characterization of a helical protein designed from 1st principles
Science
(1988) - et al.
De novo design, expression, and characterization of felix — a 4-helix bundle protein of native-like sequence
Science
(1990)
De novo design of proteins — what are the rules?
Chem Rev
Design of a novel globular protein fold with atomic-level accuracy
Science
De novo backbone scaffolds for protein design
Proteins
Protein core packing by dynamic combinatorial chemistry
J Am Chem Soc
Designing artificial enzymes by intuition and computation
Nat Chem
A tetrahedral zinc(Ii)-binding site introduced into a designed protein
Biochemistry
De novo design of mercury-binding two- and three-helical bundles
J Am Chem Soc
Retrostructural analysis of metalloproteins: application to the design of a minimal model for diiron proteins
Proc Natl Acad Sci U S A
Spectroscopic and metal-binding properties of DF3: an artificial protein able to accommodate different metal ions
J Biol Inorg Chem
Proton and metal ion-dependent assembly of a model diiron protein
Protein Sci
Oxygen reactivity of the biferrous site in the de novo designed four helix bundle peptide DFsc: nature of the ‘intermediate’ and reaction mechanism
J Am Chem Soc
An artificial di-iron oxo-protein with phenol oxidase activity
Nat Chem Biol
Comparison of the binding of cadmium(II), mercury(II), and arsenic(III) to the de novo designed peptides TRI L12C and TRI L16C
J Am Chem Soc
De novo design of a non-natural fold for an iron-sulfur protein: alpha-helical coiled-coil with a four-iron four-sulfur cluster binding site in its central core
Biochim Biophys Acta-Bioenerg
Binding of Zn-chlorin to a synthetic four-helix bundle peptide through histidine ligation
Biochemistry
Cited by (51)
Mapping interaction between big spaces; active space from protein structure and available chemical space
2022, Big Data Analytics in Chemoinformatics and Bioinformatics: with Applications to Computer-Aided Drug Design, Cancer Biology, Emerging Pathogens and Computational ToxicologySubstrate promiscuity of a de novo designed peroxidase
2021, Journal of Inorganic BiochemistryCitation Excerpt :De novo enzymes constructed from standard biotic components that both reproduce and improve upon the chemistry of their natural counterparts are of significant interest as they offer insight into the inner workings of natural enzymes while providing robust and highly versatile catalysts for a multitude of applications [1–6].
Using Evolution to Guide Protein Engineering: The Devil IS in the Details
2016, Biophysical JournalCitation Excerpt :The holy grail of protein engineering is de novo rational design of novel sequences and functions. However, success in this area has been limited to small proteins (1–3). Thus, to engineer proteins with more complex functions, researchers have developed strategies to modify naturally evolved proteins.
Designing Covalently Linked Heterodimeric Four-Helix Bundles
2016, Methods in EnzymologyAdaptive Assembly: Maximizing the Potential of a Given Functional Peptide with a Tailor-Made Protein Scaffold
2015, Chemistry and BiologyCitation Excerpt :The ultimate goal of protein engineering is to create novel proteins with desired functions. Rational and/or combinatorial approaches have allowed for the generation of functional proteins for biomedical and industrial applications (Smith and Hecht, 2011). Generating proteins from scratch, however, still remains challenging because of the enormous sequential diversity of a protein; thus de novo protein engineering has been generally performed in a stepwise manner from several components (Blaber and Lee, 2012; Verschueren et al., 2011) such as protein scaffolds (Binz et al., 2005; Gronwall and Stahl, 2009) or functional peptides (Eichler, 2008).
Frustration in biomolecules
2014, Quarterly Reviews of Biophysics