Efficient Sampling of SCHEMA Chimera Families to Identify Useful Sequence Elements

Heinzelman, Pete; Romero, Philip A.; Arnold, Frances H.

doi:10.1016/b978-0-12-394292-0.00016-3

Cited by 19 publications

(16 citation statements)

References 14 publications

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“…Machine learning methods seem extremely well suited for searching landscapes of this type. 23,56,107,677,899 Overall, this is a very important difference between natural evolution and (Experimenter-) Directed Evolution.…”

Section: Assessment Of Diversity and Its Maintenancementioning

confidence: 99%

Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

et al. 2015

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Section: Assessment Of Diversity and Its Maintenancementioning

confidence: 99%

Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

et al. 2015

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“…That protein structure can be designed computationally was established a long time ago (1) and has been demonstrated numerous times since (23,26). It is also true that reliance on prior structural data has been broadly explored, both in terms of various statistics-based methods (46,49,52,54) as well as in the creation of chimeras that fused domains or larger fragments from existing structures (64)(65)(66)(67). What is new and exciting about our results here is the marriage between the generality of our approach (i.e., its ability to design sequences for arbitrarily-defined structures) and its reliance on motif-based structural data.…”

Section: Discussionmentioning

confidence: 99%

A general-purpose protein design framework based on mining sequence-structure relationships in known protein structures

Zhou

Panaitiu

Grigoryan

2018

Preprint

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The ability to routinely design functional proteins, in a targeted manner, would have enormous implications for biomedical research and therapeutic development. Computational protein design (CPD) offers the potential to fulfill this need, and though recent years have brought considerable progress in the field, major limitations remain. Current state-of-the-art approaches to CPD aim to capture the determinants of structure from physical principles. While this has led to many successful designs, it does have strong limitations associated with inaccuracies in physical modeling, such that a robust general solution to CPD has yet to be found. Here we propose a fundamentally novel design framework-one based on identifying and applying patterns of sequence-structure compatibility found in known proteins, rather than approximating them from models of inter-atomic interactions. Specifically, we systematically decompose the target structure to be designed into structural building blocks we call TERMs (tertiary motifs) and use rapid structure search against the Protein Data Bank (PDB) to identify sequence patterns associated with each TERM from known protein structures that contain it. These results are then combined to produce a sequence-level pseudo-energy model that can score any sequence for compatibility with the target structure. This model can then be used to extract the optimal-scoring sequence via combinatorial optimization or otherwise sample the sequence space predicted to be well compatible with folding to the target. Here we carry out extensive computational analyses, showing that our method, which we dub dTERMen (design with TERM energies): 1) produces native-like sequences given native crystallographic or NMR backbones, 2) produces sequence-structure compatibility scores that correlate with thermodynamic stability, and 3) is able to predict experimental success of designed sequences generated with other methods, and 4) designs sequences that are found to fold to the desired target by structure prediction more frequently than sequences designed with an atomistic method. As an experimental validation of dTERMen, we perform a total surface redesign of Red Fluorescent Protein mCherry, marking a total of 64 residues as variable. The single sequence identified as optimal by dTERMen harbors 48 mutations relative to mCherry, but nevertheless folds, is monomeric in solution, exhibits similar stability to chemical denaturation as mCherry, and even preserves the fluorescence property. Our results strongly argue that the PDB is now sufficiently large to enable proteins to be designed by using only examples of structural motifs from unrelated proteins. This is highly significant, given that the structural database will only continue to grow, and signals the possibility of a whole host of novel data-driven CPD methods. Because such methods are likely to have orthogonal strengths relative to existing techniques, they could represent an important step towards removing remaining barriers to robust CPD.

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“…Homologous recombination can be used in the laboratory to study how primary structure controls protein function 8,9 . This approach is appealing to use for protein design because amino acid substitutions created by recombination are less disruptive than those created randomly, since the sequence blocks being swapped have already been selected by evolution for compatibility within native structures 10 .…”

Section: Whilementioning

confidence: 99%

Recombination of 2Fe-2S ferredoxins reveals differences in the inheritance of thermostability and midpoint potential

Campbell

Kahanda

Atkinson

et al. 2020

Preprint

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Homologous recombination can be used to create enzymes that exhibit distinct activities and stabilities from proteins in nature, allowing researchers to overcome component limitations in synthetic biology. To investigate how recombination affects the physical properties of an oxidoreductase that transfers electrons, we created ferredoxin (Fd) chimeras by recombining distantly-related cyanobacterial and cyanomyophage Fds that present similar midpoint potentials but distinct thermostabilities. Fd chimeras having a wide range of amino acid substitutions retained the ability to coordinate an iron-sulfur cluster, although their thermostabilities varied with the fraction of residues inherited from each parent. The midpoint potentials of chimeric Fds also varied. However, all of the synthetic Fds exhibited midpoint potentials outside of the parental protein range. Each of the chimeric Fds could also be used to build synthetic pathways that support electron transfer between Fd-NADP reductase and sulfite reductase in Escherichia coli, although the chimeric Fds varied in the expression required to support similar levels of cellular electron transfer. These results show how recombination can be used to rapidly diversify the physical properties of protein electron carriers and reveal differences in the inheritance of thermostability and electrochemical properties. Furthermore, they illustrate how electron transfer efficiencies of chimeric Fds can be rapidly evaluated using a synthetic electron transfer pathway.

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Efficient Sampling of SCHEMA Chimera Families to Identify Useful Sequence Elements

Cited by 19 publications

References 14 publications

Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

A general-purpose protein design framework based on mining sequence-structure relationships in known protein structures

Recombination of 2Fe-2S ferredoxins reveals differences in the inheritance of thermostability and midpoint potential

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