Protein engineers turned evolutionists—the quest for the optimal starting point

Trudeau, Devin L.; Tawfik, Dan S.

doi:10.1016/j.copbio.2018.12.002

Cited by 116 publications

(97 citation statements)

References 86 publications

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“… 62 Finally, the computationally designed 248-residued RA can be modified at approximately 30 positions. This signifies the genetic “plasticity” 107 , 108 of RA and echoes the fact that TIM-barrel fold is found in at least 15 families of enzymes. 109 – 111 The coupling of computational design (rational) and laboratory evolution with high-throughput screening (randomness) has proven to be an effective approach to create de novo enzyme.…”

Section: De Novo Design/laboratory Evolutionmentioning

confidence: 59%

Enabling protein-hosted organocatalytic transformations

et al. 2020

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Section: De Novo Design/laboratory Evolutionmentioning

confidence: 59%

Enabling protein-hosted organocatalytic transformations

et al. 2020

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“…The presence of these events may pinpoint loop remodelling events 19 or other conformational diversity in the family. In turn, the evolution of conformational diversity may promote new functions 8 .…”

Section: Discussionmentioning

confidence: 99%

“…With this increased coverage of natural diversity we are now better placed than ever before to leverage ancestral sequence reconstruction (ASR) to recover the ancestral portion and trace the evolutionary events that determine biological function and structure 4 . This is especially useful for protein engineering; the evolutionary record reveals essential cues for the discovery of new enzymes and resurrection of ancestral enzymes often generates enzymes with novel properties that can be exploited in biocatalysis 5,6,7,8 .…”

Section: Introductionmentioning

confidence: 99%

Engineering indel and substitution variants of diverse and ancient enzymes using Graphical Representation of Ancestral Sequence Predictions (GRASP)

Foley

Mora

Ross

et al. 2019

Preprint

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We developed Graphical Representation of Ancestral Sequence Predictions (GRASP) to infer and explore ancestral variants of protein families with more than 10,000 members. GRASP uses partial order graphs to represent homology in very large datasets, which are intractable with current inference tools and may, for example, be used to engineer proteins by identifying ancient variants of enzymes. We demonstrate that (1) across three distinct enzyme families, GRASP predicts ancestor sequences, all of which demonstrate enzymatic activity, (2) within-family insertions and deletions can be used as building blocks to support the engineering of biologically active ancestors via a new source of ancestral variation, and (3) generous inclusion of sequence data encompassing great diversity leads to less variance in ancestor sequence.GRASP is the central tool in the GRASP-suite, which is freely available at

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“…It has been previously observed that when a protein is subjected to directed evolution to attain an altered function, the stability of the resulting mutants is often compromised. [18][19][20][21] Consequently, the extent to which a protein can be engineered is often limited by how stable it is. We hypothesized that the success in engineering MjTyrRS, an enzyme derived from a thermophilic archaeon, is likely facilitated by its high structural stability.…”

mentioning

confidence: 99%

“…Work in the last two decades have provided a deep insight into how the biophysical properties of a protein influence its evolution. [18][19][20][21] It is now clear that the function-altering mutations acquired during experimental or natural evolution can often negatively impact the structural stability of a protein. Our work highlights its impact on the GCE technology, which relies on engineered aaRSs that selectively charge ncAAs of interest.…”

mentioning

confidence: 99%

Structural robustness affects the engineerability of aminoacyl-tRNA synthetases for genetic code expansion

Grasso

Yeo

Hillenbrand

et al. 2019

Preprint

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The ability to engineer the substrate specificity of natural aminoacyl-tRNA synthetase/tRNA pairs facilitates the site-specific incorporation of noncanonical amino acids (ncAAs) into proteins. The Methanocaldococcus jannaschii derived tyrosyl-tRNA synthetase (MjTyrRS)/tRNA pair has been engineered to incorporate numerous ncAAs into protein expressed in bacteria. However, it cannot be used in eukaryotic cells due to cross-reactivity with its host counterparts. The E. coli derived tyrosyl-tRNA synthetase (EcTyrRS)/tRNA pair offers a suitable alternative to this end, but a much smaller subset of ncAAs has been genetically encoded using this pair. Here we report that this discrepancy, at least partly, stems from the lower structural robustness of EcTyrRS relative to MjTyrRS. We show that engineered TyrRS mutants in general exhibit significantly lower thermostability relative to their wild-type counterparts. Derived from a thermophilic archaeon, MjTyrRS is a remarkably sturdy protein and tolerates extensive active site engineering without a catastrophic loss of stability at physiological temperature. In contrast, EcTyrRS exhibits significantly lower thermostability, rendering some of its engineered mutants insufficiently stable at physiological temperature. Our observations identify the structural robustness of an aaRS as an important factor that significantly influences how extensively it can be engineered. To overcome this limitation, we have further developed chimeras between EcTyrRS and its homolog from a thermophilic bacteria, which offer an optimal balance between thermostability and activity. We show that the chimeric bacterial TyrRSs show enhanced tolerance for destabilizing active site mutations, providing a potentially more engineerable platform for genetic code expansion.

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Protein engineers turned evolutionists—the quest for the optimal starting point

Cited by 116 publications

References 86 publications

Enabling protein-hosted organocatalytic transformations

Enabling protein-hosted organocatalytic transformations

Engineering indel and substitution variants of diverse and ancient enzymes using Graphical Representation of Ancestral Sequence Predictions (GRASP)

Structural robustness affects the engineerability of aminoacyl-tRNA synthetases for genetic code expansion

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