Design of a Fe4S4 cluster into the core of a de novo four‐helix bundle

Mancini, Joshua A.; Pike, Douglas H.; Tyryshkin, Alexei M.; Haramaty, Liti; Wang, Michael S.; Poudel, Saroj; Hecht, Michael H.; Nanda, Vikas

doi:10.1002/bab.2003

Cited by 6 publications

(4 citation statements)

References 82 publications

(97 reference statements)

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“… 54 A small number of de novo protein model systems were designed to study Fe-S clusters. 55 − 57 In those studies, the authors have one cluster per protein and therefore cannot show directionality in electron transfer reactions. In this work, we show tetrahelical bundles engineered to create different redox domains—with tailorable distances—that can drive electron transfer in one direction.…”

Section: Discussionmentioning

confidence: 99%

Tailorable Tetrahelical Bundles as a Toolkit for Redox Studies

et al. 2022

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Oxidoreductases have evolved over millions of years to perform a variety of metabolic tasks crucial for life. Understanding how these tasks are engineered relies on delivering external electron donors or acceptors to initiate electron transfer reactions. This is a challenge. Small-molecule redox reagents can act indiscriminately, poisoning the cell. Natural redox proteins are more selective, but finding the right partner can be difficult due to the limited number of redox potentials and difficulty tuning them. De novo proteins offer an alternative path. They are robust and can withstand mutations that allow for tailorable changes. They are also devoid of evolutionary artifacts and readily bind redox cofactors. However, no reliable set of engineering principles have been developed that allow for these proteins to be fine-tuned so their redox midpoint potential ( E m ) can form donor/acceptor pairs with any natural oxidoreductase. This work dissects protein-cofactor interactions that can be tuned to modulate redox potentials of acceptors and donors using a mutable de novo designed tetrahelical protein platform with iron tetrapyrrole cofactors as a test case. We show a series of engineered heme b -binding de novo proteins and quantify their resulting effect on E m . By focusing on the surface charge and buried charges, as well as cofactor placement, chemical modification, and ligation of cofactors, we are able to achieve a broad range of E m values spanning a range of 330 mV. We anticipate this work will guide the design of proteinaceous tools that can interface with natural oxidoreductases inside and outside the cell while shedding light on how natural proteins modulate E m values of bound cofactors.

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Section: Discussionmentioning

confidence: 99%

Tailorable Tetrahelical Bundles as a Toolkit for Redox Studies

et al. 2022

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“…In this respect, Kariyawasam et al [9] developed a new iron hydrogenase for the generation of secondary alcohols from the corresponding ketone. Mancini, Pike et al [10] demonstrate once again the versatility of four-helix bundles in accommodating various metal sites. In particular, they report on the successful incorporation of a Fe 4 S 4 cluster into the core of a de novo four-helix bundle, a non-natural fold for iron-sulfur clusters.…”

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confidence: 96%

Special issue (67:4): Synthetic and engineered enzymes for biocatalysis and biotransformation

Lombardi

Cheruzel

Liu

2020

Biotech and App Biochem

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Recent advances in synthetic and engineered enzymes have paved the way for unique laboratory and industrial applications. Design of artificial metalloenzymes using native or de novo scaffolds as well as repurposing of metalloenzymes and protein engineering have enabled investigators to enhance enzyme properties (e.g., thermostability, organic solvent compatibility) and expand their reaction scope. In addition, enzyme immobilization often facilitates their separation from the reaction mixture and their reusability. This special issue of Biotechnology and Applied Biochemistry highlights some recent examples encompassing the utilization of enzymes and metalloenzymes for unique biocatalytic, biosensing and biotransformation applications.

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“…Importantly, these proteins were neither selected by nature nor do they have any relationship to natural biochemical pathways. Several proteins from this “alternate universe” of sequences have been shown to possess binding and/or catalytic activities both in vitro and in vivo ( 1 , 5 , 14 – 22 ). The occurrence of enzymatic activity is especially surprising as these sequences have no relationship to natural proteins and were not explicitly designed to have catalytic activity.…”

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confidence: 99%

A de novo protein catalyzes the synthesis of semiconductor quantum dots

Spangler

Yao

Cheng

et al. 2022

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

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De novo proteins constructed from novel amino acid sequences are distinct from proteins that evolved in nature. Construct K (ConK) is a binary-patterned de novo designed protein that rescues Escherichia coli from otherwise toxic concentrations of copper. ConK was recently found to bind the cofactor PLP (pyridoxal phosphate, the active form of vitamin B 6 ). Here, we show that ConK catalyzes the desulfurization of cysteine to H 2 S, which can be used to synthesize CdS nanocrystals in solution. The CdS nanocrystals are approximately 3 nm, as measured by transmission electron microscope, with optical properties similar to those seen in chemically synthesized quantum dots. The CdS nanocrystals synthesized using ConK have slower growth rates and a different growth mechanism than those synthesized using natural biomineralization pathways. The slower growth rate yields CdS nanocrystals with two desirable properties not observed during biomineralization using natural proteins. First, CdS nanocrystals are predominantly of the zinc blende crystal phase; this is in stark contrast to natural biomineralization routes that produce a mixture of zinc blende and wurtzite phase CdS. Second, in contrast to the growth and eventual precipitation observed in natural biomineralization systems, the CdS nanocrystals produced by ConK stabilize at a final size. Future optimization of CdS nanocrystal growth using ConK—or other de novo proteins—may help to overcome the limits on nanocrystal quality typically observed from natural biomineralization by enabling the synthesis of more stable, high-quality quantum dots at room temperature.

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Design of a Fe₄S₄ cluster into the core of a de novo four‐helix bundle

Cited by 6 publications

References 82 publications

Tailorable Tetrahelical Bundles as a Toolkit for Redox Studies

Tailorable Tetrahelical Bundles as a Toolkit for Redox Studies

Special issue (67:4): Synthetic and engineered enzymes for biocatalysis and biotransformation

A de novo protein catalyzes the synthesis of semiconductor quantum dots

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