Intense Neutral Drifts Yield Robust and Evolvable Consensus Proteins

Bershtein, Shimon; Goldin, Korina; Tawfik, Dan S.

doi:10.1016/j.jmb.2008.04.024

Cited by 240 publications

(284 citation statements)

References 55 publications

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“…Experiments showed that many natural globular proteins are robust to mutations [40,53,55,202,360]. The observation that proteins with diverse primary sequences can be grouped into families with very similar structure and function gives further illustration of this robustness [361].…”

Section: Mutational Robustness Sequence-space Topology and Populatiomentioning

confidence: 94%

Biophysics of protein evolution and evolutionary protein biophysics

Sikosek

Chan

2014

J. R. Soc. Interface.

224

207

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The study of molecular evolution at the level of protein-coding genes often entails comparing large datasets of sequences to infer their evolutionary relationships. Despite the importance of a protein's structure and conformational dynamics to its function and thus its fitness, common phylogenetic methods embody minimal biophysical knowledge of proteins. To underscore the biophysical constraints on natural selection, we survey effects of protein mutations, highlighting the physical basis for marginal stability of natural globular proteins and how requirement for kinetic stability and avoidance of misfolding and misinteractions might have affected protein evolution. The biophysical underpinnings of these effects have been addressed by models with an explicit coarse-grained spatial representation of the polypeptide chain. Sequence-structure mappings based on such models are powerful conceptual tools that rationalize mutational robustness, evolvability, epistasis, promiscuous function performed by 'hidden' conformational states, resolution of adaptive conflicts and conformational switches in the evolution from one protein fold to another. Recently, protein biophysics has been applied to derive more accurate evolutionary accounts of sequence data. Methods have also been developed to exploit sequence-based evolutionary information to predict biophysical behaviours of proteins. The success of these approaches demonstrates a deep synergy between the fields of protein biophysics and protein evolution.

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Section: Mutational Robustness Sequence-space Topology and Populatiomentioning

confidence: 94%

Biophysics of protein evolution and evolutionary protein biophysics

Sikosek

Chan

2014

J. R. Soc. Interface.

224

207

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“…These apparent T M values did not correlate with soluble expression levels (see SI Appendix, Fig. S2), possibly due to the fact that many consensus mutations affect kinetic rather than thermodynamic stability (25,27). The primary outcome of the incorporated consensus mutations was therefore an increased tolerance to mutations, which enabled the directed evolution of KE59 towards higher catalytic activity.…”

Section: Resultsmentioning

confidence: 99%

“…Such consensus mutations were previously shown to promote protein evolvability (25). The sequence of the template protein of KE59 was aligned to sequences of related IGPSs (42-57% identity to KE59 design; see SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 99%

“…However, as is the case with many natural proteins, the stability of KE59 was limited, and so was its evolvability-no improved variants were observed in libraries of random mutations. Consensus mutations comprise the most easily identifiable potential of stabilizing mutations that can boost protein evolvability (25). As less than half of the predicted consensus mutations actually have a stabilizing effect (24,36), and different mutations become compensatory at different evolutionary stages (as seen in R1-R2 versus R7 and later), spiking these mutations into libraries is a more effective strategy than the use of a prestabilized starting point (20).…”

Section: Discussionmentioning

confidence: 99%

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Bridging the gaps in design methodologies by evolutionary optimization of the stability and proficiency of designed Kemp eliminase KE59

Khersonsky

Kiss

Röthlisberger

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

219

221

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Computational design is a test of our understanding of enzyme catalysis and a means of engineering novel, tailor-made enzymes. While the de novo computational design of catalytically efficient enzymes remains a challenge, designed enzymes may comprise unique starting points for further optimization by directed evolution. Directed evolution of two computationally designed Kemp eliminases, KE07 and KE70, led to low to moderately efficient enzymes (k cat ∕K m values of ≤5 × 10 4 M −1 s −1 ). Here we describe the optimization of a third design, KE59. Although KE59 was the most catalytically efficient Kemp eliminase from this design series (by k cat ∕K m , and by catalyzing the elimination of nonactivated benzisoxazoles), its impaired stability prevented its evolutionary optimization. To boost KE59's evolvability, stabilizing consensus mutations were included in the libraries throughout the directed evolution process. The libraries were also screened with less activated substrates. Sixteen rounds of mutation and selection led to >2,000-fold increase in catalytic efficiency, mainly via higher k cat values. The best KE59 variants exhibited k cat ∕K m values up to 0.6 × 10 6 M −1 s −1 , and k cat ∕k uncat values of ≤10 7 almost regardless of substrate reactivity. Biochemical, structural, and molecular dynamics (MD) simulation studies provided insights regarding the optimization of KE59. Overall, the directed evolution of three different designed Kemp eliminases, KE07, KE70, and KE59, demonstrates that computational designs are highly evolvable and can be optimized to high catalytic efficiencies.computational protein design | enzyme mimic T he endeavor of making enzyme-like catalysts spans several decades (1) with the Kemp elimination being a thoroughly explored model (2-7). In this activated model system, basecatalyzed proton elimination from carbon is concerted with the cleavage of nitrogen-oxygen bond, thus leading to the cyanophenol product (Fig. 1A). Activation of a carboxylate base catalyst by desolvation is efficiently mimicked by aprotic dipolar solvents such as acetonitrile and by various enzyme mimics. Effective alignment of the substrate and charge-dispersing interactions that stabilize the negatively charged transition state (TS) have also been achieved by various enzyme mimics that catalyze the Kemp elimination (8, 9). However, generation of a (wo)manmade active site that exhibits all these features and performs as well as natural enzymes, remains a challenge.Computational methods for predicting structure from sequence at atomic accuracy provide a new approach to enzyme engineering (10-12). The design involves two steps: (i) designing an active-site configuration that may confer efficient catalysis, (ii) computing a sequence that confers the desired configuration. Both steps are currently far from optimal, as the catalytic efficiency of designed enzymes falls far behind that of natural enzymes. Further, as with other enzyme mimics, computational designs tackle activated model systems and fail to catalyze cha...

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“…The back-to-motif mutation indicates that this substrate-determining motif could be ancestral to the GH5 family, supporting Jensen's hypothesis (40) that the spectrum of specificity in the ancestors of an enzyme family can be seen in the descendant families. Similar to back-to-ancestor mutations at nonactive sites, back-to-motif mutations within the active sites may broaden enzyme activity and also make enzymes more evolvable (41).…”

Section: Discussionmentioning

confidence: 99%

Tracing Determinants of Dual Substrate Specificity in Glycoside Hydrolase Family 5

Chen

Friedland

Pereira

et al. 2012

Journal of Biological Chemistry

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Background: Glycoside hydrolase family 5 (GH5) comprises enzymes with a wide range of activities critical for the deconstruction of lignocellulose. Results: Concurrent glucan and mannan specificity in over 70 members of GH5 can be ascribed to a conserved active site motif. Conclusion: Single domain multispecific hydrolases are widely prevalent. Significance: This finding has potential applications in improved enzyme mixture design or microbes engineered for consolidated bioprocessing of lignocellulose.

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Intense Neutral Drifts Yield Robust and Evolvable Consensus Proteins

Cited by 240 publications

References 55 publications

Biophysics of protein evolution and evolutionary protein biophysics

Biophysics of protein evolution and evolutionary protein biophysics

Bridging the gaps in design methodologies by evolutionary optimization of the stability and proficiency of designed Kemp eliminase KE59

Tracing Determinants of Dual Substrate Specificity in Glycoside Hydrolase Family 5

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