Recurring sequence-structure motifs in (βα)8-barrel proteins and experimental optimization of a chimeric protein designed based on such motifs

Wang, Jichao; Zhang, Tongchuan; Liu, Ruicun; Song, Meilin; Wang, Juncheng; Hong, Jiong; Chen, Quan; Liu, Haiyan

doi:10.1016/j.bbapap.2016.11.001

Cited by 4 publications

(4 citation statements)

References 45 publications

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“…The engineering of natural TIM barrels, both by directed evolution and design, has resulted in enzymes with altered substrate specificities, enhanced reaction rates, and even new-to-nature chemistries (6)(7)(8)(9)(10)(11)(12)(13). The de novo design of catalytic functions, however, has been challenged in part by the difficulty in programming flexible loops; the most successful approaches to date have reused loops found in nature (14,15).…”

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confidence: 99%

Tight and specific lanthanide binding in a de novo TIM barrel with a large internal cavity designed by symmetric domain fusion

Caldwell

Haydon

Piperidou

et al. 2020

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

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De novo protein design has succeeded in generating a large variety of globular proteins, but the construction of protein scaffolds with cavities that could accommodate large signaling molecules, cofactors, and substrates remains an outstanding challenge. The long, often flexible loops that form such cavities in many natural proteins are difficult to precisely program and thus challenging for computational protein design. Here we describe an alternative approach to this problem. We fused two stable proteins with C2 symmetry—a de novo designed dimeric ferredoxin fold and a de novo designed TIM barrel—such that their symmetry axes are aligned to create scaffolds with large cavities that can serve as binding pockets or enzymatic reaction chambers. The crystal structures of two such designs confirm the presence of a 420 cubic Ångström chamber defined by the top of the designed TIM barrel and the bottom of the ferredoxin dimer. We functionalized the scaffold by installing a metal-binding site consisting of four glutamate residues close to the symmetry axis. The protein binds lanthanide ions with very high affinity as demonstrated by tryptophan-enhanced terbium luminescence. This approach can be extended to other metals and cofactors, making this scaffold a modular platform for the design of binding proteins and biocatalysts.

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mentioning

confidence: 99%

Tight and specific lanthanide binding in a de novo TIM barrel with a large internal cavity designed by symmetric domain fusion

Caldwell

Haydon

Piperidou

et al. 2020

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

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“…They introduced mutations in the inter-half contacts of a β-glucosidase to obtain independent half barrels that unfold cooperatively [ 37 ]. Further, Wang et al identified physicochemical properties from a set of non-redundant TIM-barrel proteins that strongly support the existence of recurring βα and αβ motifs in this fold [ 38 ]. In addition, using a conserved αβα element as a recombination site, they created a chimeric protein from two different TIM barrels, highlighting the potential of recurring motifs as naturally optimized interfaces to engineer well-folded chimeras.…”

Section: Recreating Evolutionary Events In the Lab: Chimeragenesis And Directed Evolutionmentioning

confidence: 99%

Evolution, folding, and design of TIM barrels and related proteins

Romero‐Romero

Kordes

Michel

et al. 2021

Current Opinion in Structural Biology

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Highlights In-depth sequence analysis reveals that the protein fold universe is more evolutionarily connected than previously assumed. Short ancestral fragments are observed to have propagated to many modern proteins and hint at possible evolutionary pathways. Experimental reconstruction of such events by chimeragenesis and directed evolution allows to test evolutionary relationships. Detailed knowledge of folding landscapes helps to understand evolutionary history and improve protein engineering. The ubiquitous and versatile TIM-barrel fold is a model system to explore evolution, folding, and design.

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“…Through directed evolution approach, the foldability of the initially designed proteins was further optimized, demonstrating the ability of this system to correct the original problematic designs as an effective complement to the rational design approach. Wang et al also showed that this system can be applied to optimize the folding of a designed chimeric (βα) 8-barrel protein (TIM-barrel protein) (Wang et al 2017b). Through six rounds of evolution, the half melting temperature (T m ) of the isolated variant that led to the highest ampicillin resistance has been improved by more than 20 °C.…”

Section: β-Lactamase Tripartite Reportermentioning

confidence: 99%

Selection and screening strategies in directed evolution to improve protein stability

Ren

Wen

Mencius

et al. 2019

Bioresour. Bioprocess.

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Protein stability is not only fundamental for experimental, industrial, and therapeutic applications, but is also the baseline for evolving novel protein functions. For decades, stability engineering armed with directed evolution has continued its rapid development and inevitably poses challenges. Generally, in directed evolution, establishing a reliable link between a genotype and any interpretable phenotype is more challenging than diversifying genetic libraries. Consequently, we set forth in a small picture to emphasize the screening or selection techniques in protein stability-directed evolution to secure the link. For a more systematic review, two main branches of these techniques, namely cellular or cell-free display and stability biosensors, are expounded with informative examples.

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Recurring sequence-structure motifs in (βα)8-barrel proteins and experimental optimization of a chimeric protein designed based on such motifs

Cited by 4 publications

References 45 publications

Tight and specific lanthanide binding in a de novo TIM barrel with a large internal cavity designed by symmetric domain fusion

Tight and specific lanthanide binding in a de novo TIM barrel with a large internal cavity designed by symmetric domain fusion

Evolution, folding, and design of TIM barrels and related proteins

Selection and screening strategies in directed evolution to improve protein stability

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