4 publications
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Metal-Mediated Protein Assembly Using a Genetically Incorporated Metal-Chelating Amino Acid
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Biomacromolecules 2020, 21, 5021-5028, 10.1021/acs.biomac.0c01194
Many natural proteins function in oligomeric forms, which are critical for their sophisticated functions. The construction of protein assemblies has great potential for biosensors, enzyme catalysis, and biomedical applications. In designing protein assemblies, a critical process is to create protein–protein interaction (PPI) networks at defined sites of a target protein. Although a few methods are available for this purpose, most of them are dependent on existing PPIs of natural proteins to some extent. In this report, a metal-chelating amino acid, 2,2′-bipyridylalanine (BPA), was genetically introduced into defined sites of a monomeric protein and used to form protein oligomers. Depending on the number of BPAs introduced into the protein and the species of metal ions (Ni2+ and Cu2+), dimers or oligomers with different oligomerization patterns were formed by complexation with a metal ion. Oligomer sizes could also be controlled by incorporating two BPAs at different locations with varied angles to the center of the protein. When three BPAs were introduced, the monomeric protein formed a large complex with Ni2+. In addition, when Cu2+ was used for complex formation with the protein containing two BPAs, a linear complex was formed. The method proposed in this report is technically simple and generally applicable to various proteins with interesting functions. Therefore, this method would be useful for the design and construction of functional protein assemblies.
Ligand type: BipyridineHost protein: Maltose-binding protein (MBP)Anchoring strategy: DativeOptimization: ---Reaction: ---Max TON: ---ee: ---PDB: ---Notes: ---
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Rare Earth Metal Ions as Probes of Calcium Binding Sites in Proteins: Neodynium Acceleration of the Activation of Trypsinogen
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J. Biol. Chem. 1970, n/a
The rate of activation of the conversion of trypsinogen to trypsin has been found to be greatly accelerated by the neodymium(III) ion. The similarity of this process to the calcium(II) ion activation suggests that both metal ions bind at identical sites in trypsinogen. The rate of activation in the presence of the neodymium ion is much greater than that of the calcium ion, probably reflecting the increased stability constant of the neodymium-protein complex. In contrast to the calcium ion, however, neodymium(III) can be scrutinized by a variety of spectral and magnetic techniques which should reveal information concerning the calcium ion binding sites in proteins. Since the chemistry and the range of sires of the rare earth metal ions are so similar to that of the calcium ion, it is suggested that generally these ions should make good replacement ions for probing the calcium ion binding sites of proteins and enzymes.
Metal: NdLigand type: Amino acidHost protein: TrypsinAnchoring strategy: Metal substitutionOptimization: ---Notes: PMID 5484822
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Selection and Evolution of Enzymes from a Partially Randomized Non-Catalytic Scaffold
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Nature 2007, 448, 828-831, 10.1038/nature06032
Enzymes are exceptional catalysts that facilitate a wide variety of reactions under mild conditions, achieving high rate-enhancements with excellent chemo-, regio- and stereoselectivities. There is considerable interest in developing new enzymes for the synthesis of chemicals and pharmaceuticals1,2,3 and as tools for molecular biology. Methods have been developed for modifying and improving existing enzymes through screening, selection and directed evolution4,5. However, the design and evolution of truly novel enzymes has relied on extensive knowledge of the mechanism of the reaction6,7,8,9,10. Here we show that genuinely new enzymatic activities can be created de novo without the need for prior mechanistic information by selection from a naive protein library of very high diversity, with product formation as the sole selection criterion. We used messenger RNA display, in which proteins are covalently linked to their encoding mRNA11, to select for functional proteins from an in vitro translated protein library of >1012independent sequences without the constraints imposed by any in vivo step. This technique has been used to evolve new peptides and proteins that can bind a specific ligand12,13,14,15,16,17,18, from both random-sequence libraries12,14,15,16 and libraries based on a known protein fold17,18. We now describe the isolation of novel RNA ligases from a library that is based on a zinc finger scaffold18,19, followed by in vitro directed evolution to further optimize these enzymes. The resulting ligases exhibit multiple turnover with rate enhancements of more than two-million-fold.
Metal: ZnLigand type: Amino acidHost protein: Human retinoid-X-receptor (hRXRa)Anchoring strategy: DativeOptimization: GeneticNotes: ---
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Semisynthesis of Bipyridyl-Alanine Cytochrome c Mutants: Novel Proteins with Enhanced Electron-Transfer Properties
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J. Am. Chem. Soc. 1993, 115, 8455-8456, 10.1021/ja00071a068
n/a
Notes: No catalysis