Engineering a zinc binding site into the de novo designed protein DS119 with a βαβ structure

Zhu, Cheng; Liang, Huanhuan; Lai, Luhua

doi:10.1007/s13238-011-1121-3

Cited by 11 publications

(18 citation statements)

References 41 publications

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“…Residues that are bulky and hydrophobic, such as Tyr, Phe, Thr, Ile, Val, and Trp, seem to have a higher propensity towards the formation of β-sheet structures. Despite these challenges, there are several successes in the design of β-sheet structures [84–89] and related scaffolds including mixed α/β structures [90–93], γ turns [94], β-hairpin peptides [95–98], and small peptides with loop region metal clusters [99,100]. There is also limited success in the de novo design of β-structured scaffolds for functional metalloproteins; the design of a redox-active rubredoxin mimic is an example of one of these rare β-sheet metalloproteins [101].…”

Section: Metalloprotein Design Approaches and Scaffold Choicesmentioning

confidence: 99%

Designing functional metalloproteins: From structural to catalytic metal sites

Zastrow

Pecoraro

2013

Coordination Chemistry Reviews

116

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Metalloenzymes efficiently catalyze some of the most important and difficult reactions in nature. For many years, coordination chemists have effectively used small molecule models to understand these systems. More recently, protein design has been shown to be an effective approach for mimicking metal coordination environments. Since the first designed proteins were reported, much success has been seen for incorporating metal sites into proteins and attaining the desired coordination environment but until recently, this has been with a lack of significant catalytic activity. Now there are examples of designed metalloproteins that, although not yet reaching the activity of native enzymes, are considerably closer. In this review, we highlight work leading up to the design of a small metalloprotein containing two metal sites, one for structural stability (HgS3) and the other a separate catalytic zinc site to mimic carbonic anhydrase activity (ZnN3O). The first section will describe previous studies that allowed for a high affinity thiolate site that binds heavy metals in a way that stabilizes three-stranded coiled coils. The second section will examine ways of preparing histidine rich environments that lead to metal based hydrolytic catalysts. We will also discuss other recent examples of the design of structural metal sites and functional metalloenzymes. Our work demonstrates that attaining the proper first coordination geometry of a metal site can lead to a significant fraction of catalytic activity, apparently independent of the type of secondary structure of the surrounding protein environment. We are now in a position to begin to meet the challenge of building a metalloenzyme systematically from the bottom-up by engineering and analyzing interactions directly around the metal site and beyond.

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Section: Metalloprotein Design Approaches and Scaffold Choicesmentioning

confidence: 99%

Designing functional metalloproteins: From structural to catalytic metal sites

Zastrow

Pecoraro

2013

Coordination Chemistry Reviews

116

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“…The genes encoding Tbab proteins were synthesized and cloned into the BamHI and XhoI sites of pGEX 4T‐1 vectors (Invitrogen, Gaithersburg, MD) as reported before . Point mutations were generated with site‐directed mutagenesis kits (Saibaisheng, Beijing, China) according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

“…All proteins were expressed and purified as previously described . Briefly, they were expressed in Escherichia coli as a GST‐fusion protein (GST: glutathione S‐transferase) and purified on a GST‐affinity column (GE Healthcare, Piscataway, NJ) and then by reversed‐phase HPLC.…”

Section: Methodsmentioning

confidence: 99%

“…By far a diverse range of protein folds has been designed from scratch, including Top7 (an α/β protein), four helix bundles and DS119 (a βαβ motif) . Top7 has been engineered to display conformation‐specific HIV‐1 epitopes; four helix bundles and DS119 have been designed to incorporate metal‐binding sites, which indicates their versatility for protein engineering purpose. Importantly, de novo designed scaffolds usually exhibit high thermal stability, making them suitable for anchoring functional residues …”

Section: Introductionmentioning

confidence: 99%

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Rational design of TNFα binding proteins based on the de novo designed protein DS119

et al. 2016

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De novo protein design offers templates for engineering tailor-made protein functions and orthogonal protein interaction networks for synthetic biology research. Various computational methods have been developed to introduce functional sites in known protein structures. De novo designed protein scaffolds provide further opportunities for functional protein design. Here we demonstrate the rational design of novel tumor necrosis factor alpha (TNFa) binding proteins using a home-made grafting program AutoMatch. We grafted three key residues from a virus 2L protein to a de novo designed small protein, DS119, with consideration of backbone flexibility. The designed proteins bind to TNFa with micromolar affinities. We further optimized the interface residues with RosettaDesign and significantly improved the binding capacity of one protein Tbab1-4. These designed proteins inhibit the activity of TNFa in cellular luciferase assays. Our work illustrates the potential application of the de novo designed protein DS119 in protein engineering, biomedical research, and protein sequence-structure-function studies.

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“…[108] In an alternative approach, bab structures computationally designed were shown to bind mononuclear zinc ions. [109] Quinone binding to these novel proteins was shown in one example, where a three-helix bundle was designed with a cysteine to link 2,6-dimethylbenzoquinone (DMBQ). [110] These examples provide hope for incorporating metal clusters and various electron acceptors such as quinones in photocatalytic electron transfer in proteins through de novo engineering approach.…”

Section: Modified Bacterioferritin As a Photoactive Reaction Centrementioning

confidence: 99%

Perspectives for Photobiology in Molecular Solar Fuels

Hingorani

Hillier

2012

Aust. J. Chem.

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This paper presents an overview of the prospects for bio-solar energy conversion. The Global Artificial Photosynthesis meeting at Lord Howe Island (14–18 August 2011) underscored the dependence that the world has placed on non-renewable energy supplies, particularly for transport fuels, and highlighted the potential of solar energy. Biology has used solar energy for free energy gain to drive chemical reactions for billions of years. The principal conduits for energy conversion on earth are photosynthetic reaction centres – but can they be harnessed, copied and emulated? In this communication, we initially discuss algal-based biofuels before investigating bio-inspired solar energy conversion in artificial and engineered systems. We show that the basic design and engineering principles for assembling photocatalytic proteins can be used to assemble nanocatalysts for solar fuel production.

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Engineering a zinc binding site into the de novo designed protein DS119 with a βαβ structure

Cited by 11 publications

References 41 publications

Designing functional metalloproteins: From structural to catalytic metal sites

Designing functional metalloproteins: From structural to catalytic metal sites

Rational design of TNFα binding proteins based on the de novo designed protein DS119

Perspectives for Photobiology in Molecular Solar Fuels

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