scholarly journals Computational design of a modular protein sense/response system

2019 ◽  
Author(s):  
Anum A. Glasgow ◽  
Yao-Ming Huang ◽  
Daniel J. Mandell ◽  
Michael Thompson ◽  
Ryan Ritterson ◽  
...  
Keyword(s):  
De Novo ◽  
Protein Structures ◽  
Computational Design ◽  
Metabolic Intermediate ◽  
Signaling Systems ◽  
Engineering Strategy ◽  
Valuable Compounds ◽  
Protein Sensors ◽  
New Protein

ABSTRACTSensing and responding to signals is a fundamental ability of living systems, but despite remarkable progress in computational design of new protein structures, there is no general approach for engineering arbitrary new protein sensors. Here we describe a generalizable computational strategy for designing sensor/actuator proteins by building binding sites de novo into heterodimeric protein-protein interfaces and coupling ligand sensing to modular actuation via split reporters. Using this approach, we designed protein sensors that respond to farnesyl pyrophosphate, a metabolic intermediate in the production of valuable compounds. The sensors are functional in vitro and in cells, and the crystal structure of the engineered binding site matches the design model with atomic accuracy. Our computational design strategy opens broad avenues to link biological outputs to new signals.One Sentence SummaryAn engineering strategy to design modular synthetic signaling systems that respond to new small molecule inputs.

scholarly journals Computational design of a modular protein sense-response system

Science ◽  
2019 ◽  
Vol 366 (6468) ◽  
pp. 1024-1028 ◽  
Author(s):  
Anum A. Glasgow ◽  
Yao-Ming Huang ◽  
Daniel J. Mandell ◽  
Michael Thompson ◽  
Ryan Ritterson ◽  
...  
Keyword(s):  
De Novo ◽  
Protein Structures ◽  
Computational Design ◽  
Metabolic Intermediate ◽  
Protein Interfaces ◽  
Substantial Progress ◽  
Valuable Compounds ◽  
Protein Sensors ◽  
New Protein

Sensing and responding to signals is a fundamental ability of living systems, but despite substantial progress in the computational design of new protein structures, there is no general approach for engineering arbitrary new protein sensors. Here, we describe a generalizable computational strategy for designing sensor-actuator proteins by building binding sites de novo into heterodimeric protein-protein interfaces and coupling ligand sensing to modular actuation through split reporters. Using this approach, we designed protein sensors that respond to farnesyl pyrophosphate, a metabolic intermediate in the production of valuable compounds. The sensors are functional in vitro and in cells, and the crystal structure of the engineered binding site closely matches the design model. Our computational design strategy opens broad avenues to link biological outputs to new signals.

scholarly journals De novo design of self-assembling helical protein filaments

Science ◽  
2018 ◽  
Vol 362 (6415) ◽  
pp. 705-709 ◽  
Author(s):  
Hao Shen ◽  
Jorge A. Fallas ◽  
Eric Lynch ◽  
William Sheffler ◽  
Bradley Parry ◽  
...  
Keyword(s):  
De Novo ◽  
Computational Design ◽  
Building Blocks ◽  
Repeat Units ◽  
Self Assembling ◽  
Wide Range ◽  
Helical Protein ◽  
Monomeric Proteins

We describe a general computational approach to designing self-assembling helical filaments from monomeric proteins and use this approach to design proteins that assemble into micrometer-scale filaments with a wide range of geometries in vivo and in vitro. Cryo–electron microscopy structures of six designs are close to the computational design models. The filament building blocks are idealized repeat proteins, and thus the diameter of the filaments can be systematically tuned by varying the number of repeat units. The assembly and disassembly of the filaments can be controlled by engineered anchor and capping units built from monomers lacking one of the interaction surfaces. The ability to generate dynamic, highly ordered structures that span micrometers from protein monomers opens up possibilities for the fabrication of new multiscale metamaterials.

scholarly journals De Novo Protein Design of Photochemical Reaction Centers

2021 ◽  
Author(s):  
Nathan Ennist ◽  
Zhenyu Zhao ◽  
Steven Stayrook ◽  
Bohdana Discher ◽  
P Leslie 'Les' Dutton ◽  
...  
Keyword(s):  
Reaction Center ◽  
Charge Separation ◽  
Rational Design ◽  
De Novo ◽  
Metal Cluster ◽  
Protein Structures ◽  
Essential Elements ◽  
Reaction Centers ◽  
Transient Absorption Spectroscopy

Abstract Natural photosynthetic protein complexes capture sunlight to power the energetic catalysis that supports life on Earth. Yet these natural protein structures carry an evolutionary legacy of complexity and fragility that encumbers protein reengineering efforts and obfuscates the underlying design rules for light-driven charge separation. De novo development of a simplified photosynthetic reaction center protein can clarify practical engineering principles needed to build new enzymes for efficient solar-to-fuel energy conversion. Here we report the rational design, X-ray crystal structure, and electron transfer activity of a multi-cofactor protein that incorporates essential elements of photosynthetic reaction centers. This highly stable, modular artificial protein framework can be reconstituted in vitro with interchangeable redox centers for nanometer-scale photochemical charge separation. Transient absorption spectroscopy demonstrates Photosystem II-like tyrosine and metal cluster oxidation, and we measure charge separation lifetimes exceeding 100 ms, ideal for light-activated catalysis. This de novo-designed reaction center builds upon engineering guidelines established for charge separation in earlier synthetic photochemical triads and modified natural proteins, and it shows how synthetic biology may lead to a new generation of genetically encoded, light-powered catalysts for solar fuel production.

scholarly journals NAD+-Dependent Deacetylase Hst1p Controls Biosynthesis and Cellular NAD+ Levels in Saccharomyces cerevisiae

2003 ◽  
Vol 23 (19) ◽  
pp. 7044-7054 ◽  
Author(s):  
Antonio Bedalov ◽  
Maki Hirao ◽  
Jeffrey Posakony ◽  
Melisa Nelson ◽  
Julian A. Simon
Keyword(s):  
De Novo ◽  
Critical Role ◽  
Salvage Pathway ◽  
De Novo Synthesis ◽  
Array Analysis ◽  
Binding Partner ◽  
Dependent Protein ◽  
New Protein

ABSTRACT Nicotine adenine dinucleotide (NAD+) performs key roles in electron transport reactions, as a substrate for poly(ADP-ribose) polymerase and NAD+-dependent protein deacetylases. In the latter two processes, NAD+ is consumed and converted to ADP-ribose and nicotinamide. NAD+ levels can be maintained by regeneration of NAD+ from nicotinamide via a salvage pathway or by de novo synthesis of NAD+ from tryptophan. Both pathways are conserved from yeast to humans. We describe a critical role of the NAD+-dependent deacetylase Hst1p as a sensor of NAD+ levels and regulator of NAD+ biosynthesis. Using transcript arrays, we show that low NAD+ states specifically induce the de novo NAD+ biosynthesis genes while the genes in the salvage pathway remain unaffected. The NAD+-dependent deacetylase activity of Hst1p represses de novo NAD+ biosynthesis genes in the absence of new protein synthesis, suggesting a direct effect. The known Hst1p binding partner, Sum1p, is present at promoters of highly inducible NAD+ biosynthesis genes. The removal of HST1-mediated repression of the NAD+ de novo biosynthesis pathway leads to increased cellular NAD+ levels. Transcript array analysis shows that reduction in cellular NAD+ levels preferentially affects Hst1p-regulated genes in comparison to genes regulated with other NAD+-dependent deacetylases (Sir2p, Hst2p, Hst3p, and Hst4p). In vitro experiments demonstrate that Hst1p has relatively low affinity toward NAD+ in comparison to other NAD+-dependent enzymes. These findings suggest that Hst1p serves as a cellular NAD+ sensor that monitors and regulates cellular NAD+ levels.

scholarly journals Computational design of closely related proteins that adopt two well-defined but structurally divergent folds

2020 ◽  
Vol 117 (13) ◽  
pp. 7208-7215 ◽  
Author(s):  
Kathy Y. Wei ◽  
Danai Moschidi ◽  
Matthew J. Bick ◽  
Santrupti Nerli ◽  
Andrew C. McShan ◽  
...  
Keyword(s):  
Large Scale ◽  
De Novo ◽  
Conformational Transition ◽  
Protein Structures ◽  
Computational Design ◽  
Spectroscopic Characterization ◽  
De Novo Design ◽  
Viral Fusion ◽  
Naturally Occurring ◽  
Related Proteins

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 for de novo design of proteins that fold to a single state with a deep free-energy minimum [P.-S. Huang, S. E. Boyken, D. Baker, Nature 537, 320–327 (2016)], and to reengineer natural proteins to alter their dynamics [J. A. Davey, A. M. Damry, N. K. Goto, R. A. Chica, Nat. Chem. Biol. 13, 1280–1285 (2017)] or fold [P. A. Alexander, Y. He, Y. Chen, J. Orban, P. N. Bryan, Proc. Natl. Acad. Sci. U.S.A. 106, 21149–21154 (2009)], the de novo design of closely related sequences which adopt well-defined but structurally divergent structures remains an outstanding challenge. We designed 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)—reminiscent of the conformational transition of viral fusion proteins. Crystallographic and NMR spectroscopic characterization shows that both the short- and long-state sequences fold as designed. We sought to design bistable sequences for which both states are accessible, and obtained a single designed protein sequence that populates either the short state or the long state depending on the measurement conditions. The design of sequences which are poised to adopt two very different conformations sets the stage for creating large-scale conformational switches between structurally divergent forms.

De novo design of ACE2 protein decoys to neutralize SARS-CoV-2

2020 ◽  
Author(s):  
Thomas W. Linsky ◽  
Renan Vergara ◽  
Nuria Codina ◽  
Jorgen W. Nelson ◽  
Matthew J. Walker ◽  
...  
Keyword(s):  
De Novo ◽  
Rapid Development ◽  
Computational Design ◽  
Design Strategy ◽  
De Novo Design ◽  
Surface Proteins ◽  
Receptor Binding Domain ◽  
Angiotensin Converting Enzyme 2 ◽  
Designed Proteins

AbstractThere is an urgent need for the ability to rapidly develop effective countermeasures for emerging biological threats, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a generalized computational design strategy to rapidly engineer de novo proteins that precisely recapitulate the protein surface targeted by biological agents, like viruses, to gain entry into cells. The designed proteins act as decoys that block cellular entry and aim to be resilient to viral mutational escape. Using our novel platform, in less than ten weeks, we engineered, validated, and optimized de novo protein decoys of human angiotensin-converting enzyme 2 (hACE2), the membrane-associated protein that SARS-CoV-2 exploits to infect cells. Our optimized designs are hyperstable de novo proteins (∼18-37 kDa), have high affinity for the SARS-CoV-2 receptor binding domain (RBD) and can potently inhibit the virus infection and replication in vitro. Future refinements to our strategy can enable the rapid development of other therapeutic de novo protein decoys, not limited to neutralizing viruses, but to combat any agent that explicitly interacts with cell surface proteins to cause disease.

scholarly journals Fragment-based computational design of antibodies targeting structured epitopes

2021 ◽  
Author(s):  
Mauricio Aguilar Rangel ◽  
Alice Bedwell ◽  
Elisa Costanzi ◽  
Stefano Ricagno ◽  
Judith Frydman ◽  
...  
Keyword(s):  
De Novo ◽  
Computational Design ◽  
Computational Procedure ◽  
Affinity Maturation ◽  
Combinatorial Design ◽  
Starting Point ◽  
Antibody Discovery ◽  
Single Domain Antibodies ◽  
Rapid Generation

De novo design methods hold the promise of reducing the time and cost of antibody discovery, while enabling the facile and precise targeting of specific epitopes. Here we describe a fragment-based method for the combinatorial design of antibody binding loops and their grafting onto antibody scaffolds. We designed and tested six single-domain antibodies targeting different epitopes on three antigens, including the receptor-binding domain of the SARS-CoV-2 spike protein. Biophysical characterisation showed that all designs are highly stable, and bind their intended targets with affinities in the nanomolar range without any in vitro affinity maturation. We further show that a high-resolution input antigen structure is not required, as our method yields similar predictions when the input is a crystal structure or a computer-generated model. This computational procedure, which readily runs on a laptop, provides the starting point for the rapid generation of lead antibodies binding to pre-selected epitopes.

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

2019 ◽  
Author(s):  
Kathy Y. Wei ◽  
Danai Moschidi ◽  
Matthew J. Bick ◽  
Santrupti Nerli ◽  
Andrew C. McShan ◽  
...  
Keyword(s):  
Large Scale ◽  
De Novo ◽  
Conformational Transition ◽  
Protein Structures ◽  
Computational Design ◽  
Spectroscopic Characterization ◽  
De Novo Design ◽  
Viral Fusion ◽  
Naturally Occurring ◽  
Viral Fusion Proteins

AbstractThe 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) -- reminiscent of the conformational transition of viral fusion proteins (Ivanovic et al., 2013; Podbilewicz, 2014; Skehel and Wiley, 2000). Crystallographic and NMR spectroscopic characterization show that both the short and long state sequences fold as designed. We sought to design bistable sequences for which both states are accessible, and obtained a single designed protein sequence that populates either the short state or the long state depending on the measurement conditions. The design of sequences which are poised to adopt two very different conformations sets the stage for creating large scale conformational switches between structurally divergent forms.

Dissecting the stability determinants of a challenging de novo protein fold using massively parallel design and experimentation

2021 ◽  
Author(s):  
Tae-Eun Kim ◽  
Kotaro Tsuboyama ◽  
Scott Houliston ◽  
Cydney M. Martell ◽  
Claire M. Phoumyvong ◽  
...  
Keyword(s):  
Protein Design ◽  
Large Scale ◽  
De Novo ◽  
Protein Structures ◽  
Design Problems ◽  
Hydrogen Deuterium ◽  
The Stability ◽  
Universal Stability ◽  
Scale Design ◽  
New Protein

Designing entirely new protein structures remains challenging because we do not fully understand the biophysical determinants of folding stability. Yet some protein folds are easier to design than others. Previous work identified the 43-residue αββ&#945 fold as especially challenging: the best designs had only a 2% success rate, compared to 39-87% success for other simple folds (1). This suggested the αββ&#945 fold would be a useful model system for gaining a deeper understanding of folding stability determinants and for testing new protein design methods. Here, we designed over ten thousand new αββ&#945 proteins and found over three thousand of them to fold into stable structures using a high-throughput protease-based assay. Nuclear magnetic resonance, hydrogen-deuterium exchange, circular dichroism, deep mutational scanning, and scrambled sequence control experiments indicated that our stable designs fold into their designed αββ&#945 structures with exceptional stability for their small size. Our large dataset enabled us to quantify the influence of universal stability determinants including nonpolar burial, helix capping, and buried unsatisfied polar atoms, as well as stability determinants unique to the αββ&#945 topology. Our work demonstrates how large-scale design and test cycles can solve challenging design problems while illuminating the biophysical determinants of folding.

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