De novo design of bioactive protein switches

Langan, Robert A.; Boyken, Scott E.; Ng, Andrew H.; Samson, Jennifer A.; Dods, Galen; Westbrook, Alexandra; Nguyen, Taylor H.; Lajoie, Marc J.; Chen, Zibo; Berger, Stephanie; Mulligan, Vikram Khipple; Dueber, John E.; Novak, Walter R. P.; El-Samad, Hana; Baker, David

doi:10.1038/s41586-019-1432-8

Cited by 221 publications

(185 citation statements)

References 46 publications

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“…Specifically, the field is still lacking computational methods that will enable users to reliably design their system of choice without going through multiple time-consuming DBT cycles before arriving at the desired solution. Recent studies are emerging to tackle this challenge and to devise such methods for diverse RNA-, DNA-and protein-based applications, with varying degrees of success [1][2][3] . Perhaps the best known methods are the Cello algorithm and RBS calculator, which are limited to bacterial chassis 4,5 at the present time.…”

Section: Introductionmentioning

confidence: 99%

Overcoming the design, build, test (DBT) bottleneck for synthesis of nonrepetitive protein-RNA binding cassettes for RNA applications

Katz

Tripto

Goldberg

et al. 2019

Preprint

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The design-build-test (DBT) cycle in synthetic biology is considered to be a major bottleneck for progress in the field. The emergence of high-throughput experimental techniques, such as oligo libraries (OLs), combined with machine learning (ML) algorithms, provide the ingredients for a potential "big-data" solution that can generate a sufficient predictive capability to overcome the DBT bottleneck. In this work, we apply the OL-ML approach to the design of RNA cassettes used in gene editing and RNA tracking systems. RNA cassettes are typically made of repetitive hairpins, therefore hindering their retention, synthesis, and functionality. Here, we carried out a highthroughput OL-based experiment to generate thousands of new binding sites for the phage coat proteins of bacteriophages MS2 (MCP), PP7 (PCP), and Qβ (QCP). We then applied a neural network to vastly expand this space of binding sites to millions of additional predicted sites, which allowed us to identify the structural and sequence features that are critical for the binding of each RBP. To verify our approach, we designed new non-repetitive binding site cassettes and tested their functionality in U2OS mammalian cells. We found that all our cassettes exhibited multiple trackable puncta. Additionally, we designed and verified two additional cassettes, the first containing sites that can bind both PCP and QCP, and the second with sites that can bind either MCP or QCP, allowing for an additional orthogonal channel. Consequently, we provide the scientific community with a novel resource for rapidly creating functional non-repetitive binding site cassettes using one or more of three phage coat proteins with a variety of binding affinities for any application spanning bacteria to mammalian cells.

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Section: Introductionmentioning

confidence: 99%

Overcoming the design, build, test (DBT) bottleneck for synthesis of nonrepetitive protein-RNA binding cassettes for RNA applications

Katz

Tripto

Goldberg

et al. 2019

Preprint

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“…The design of novel functional proteins is a fundamental test to our understanding of protein structure and function, and has enormous potential in multiple arenas in basic biology and biotechnology (7)(8)(9)(10)(11)(12)(13)(14)(15)17). Nevertheless, the majority of studies where function was the main objective, existing proteins were repurposed and used as templates to install functional sites (7,(17)(18)(19)(20).…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, the design of de novo proteins encoding biochemical functions is lagging far behind (6). Nonetheless, successes to date illustrate the potential of de novo design to transform multiple areas of biology and biotechnology, including the design of vaccine candidates (7-9), protein-based lead drugs (10), antivirals (11), pH-responsive carriers (12) and others (13)(14)(15).A widely used approach to design functional proteins is to transplant functional sites from their native context to heterologous proteins derived from the natural protein repertoire or de novo designed structures (16)(17)(18)(19). Commonly, we refer to this two-step approach, consisting of first selecting or building a stable, functionless scaffold, which subsequently serves as template for grafting, as a 'top-down' approach ( Fig.…”

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confidence: 99%

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confidence: 99%

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A bottom-up approach for thede novodesign of functional proteins

Yang

Sesterhenn

Bonet

et al. 2020

Preprint

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De novo protein design has enabled the creation of novel protein structures. To design novel functional proteins, state-of-the-art approaches use natural proteins or first design protein scaffolds that subsequently serve as templates for the transplantation of functional motifs. In these approaches, the templates are function-agnostic and motifs have been limited to those with regular secondary structure. Here, we present a bottom-up approach to build de novo proteins tailored to structurally complex functional motifs. We applied a bottom-up strategy to design scaffolds for four different binding motifs, including one bi-functionalized protein with two motifs. The de novo proteins were functional as biosensors to quantify epitope-specific antibody responses and as orthogonal ligands to activate a signaling pathway in engineered mammalian cells. Altogether, we present a versatile strategy for the bottom-up design of functional proteins, applicable to a wide range of functional protein design challenges.De novo protein design has emerged as a powerful approach to expand the natural protein repertoire (1-5). The majority of previous studies have been focused on structural accuracy of computational models relative to experimentally determined structures and thermodynamic stability of the designs (1-5). In contrast, the design of de novo proteins encoding biochemical functions is lagging far behind (6). Nonetheless, successes to date illustrate the potential of de novo design to transform multiple areas of biology and biotechnology, including the design of vaccine candidates (7-9), protein-based lead drugs (10), antivirals (11), pH-responsive carriers (12) and others (13)(14)(15).A widely used approach to design functional proteins is to transplant functional sites from their native context to heterologous proteins derived from the natural protein repertoire or de novo designed structures (16)(17)(18)(19). Commonly, we refer to this two-step approach, consisting of first selecting or building a stable, functionless scaffold, which subsequently serves as template for grafting, as a 'top-down' approach ( Fig. 1a).There are several important limitations of 'top-down' approaches for functional protein design.An essential prerequisite is the availability of template structures in the natural repertoire with enough local structural similarity to allow grafting of the functional site. Consequently, with a few exceptions (20), grafting has been largely limited to single, regular secondary structures that are frequently found in natural proteins (11,17,19). However, the vast majority of functional sites are not contained in single, regular helical segments, but rather are composed of multiple and often irregular structural segments that are stabilized by the overall protein structure (21-23).Similarly, most de novo proteins are built with a high content of regular secondary structures, high contact order and minimal loops (5). While these proteins generally are thermodynamically very stable, de novo proteins designed in a 'fun...

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“…In other cases, the engineered proteins have two well-defined states that are quite similar, for example the dynamic switching of DANCER proteins primarily involves a single tryptophan residue (Davey et al, 2017) , and the Rocker channel has two symmetrically related states that are structurally identical (Joh et al, 2017) . Finally, designed protein pH and peptide (Langan et al, 2019) dependent switches involve transitions between a single well-defined state and less well ordered states. Overall, in most previous work, the differences in conformation have either been relatively small, involved changes in oligomerization state, or only one of the two states has been well-defined.…”

Section: Introductionmentioning

confidence: 99%

Computational design of a protein family that adopts two well-defined and structurally divergent de novo folds

Wei

Moschidi

Bick

et al. 2019

Preprint

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The plasticity of naturally occurring protein structures, which can change shape considerably in response to changes in environmental conditions, is critical to biological function. While computational methods have been used to de novo design proteins that fold to a single state with a deep free energy minima (Huang et al., 2016) , and to reengineer natural proteins to alter their dynamics (Davey et al., 2017) or fold (Alexander et al., 2009) , the de novo design of closely related sequences which adopt well-defined, but structurally divergent structures remains an outstanding challenge. Here, we design closely related sequences (over 94% identity) that can adopt two very different homotrimeric helical bundle conformations -one short (~66 Å height) and the other long (~100 Å height) -

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De novo design of bioactive protein switches

Cited by 221 publications

References 46 publications

Overcoming the design, build, test (DBT) bottleneck for synthesis of nonrepetitive protein-RNA binding cassettes for RNA applications

Overcoming the design, build, test (DBT) bottleneck for synthesis of nonrepetitive protein-RNA binding cassettes for RNA applications

A bottom-up approach for thede novodesign of functional proteins

Computational design of a protein family that adopts two well-defined and structurally divergent de novo folds

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