Method for Enzyme Design with Genetically Encoded Unnatural Amino Acids

Hu, Cheng; Wang, J

doi:10.1016/bs.mie.2016.06.005

Cited by 6 publications

(5 citation statements)

References 55 publications

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“…And yet, one can only imagine the multitude of structures and functions available to proteins if a wider array of non-coded amino acids was exploited. While recent advances in molecular biology have allowed for the controlled incorporation of a myriad of different amino acids within proteins using amber codons or post-translational modification [1–3], the most straightforward method of incorporating alternate amino acids in protein engineering is through solid-phase synthesis [4–9]. The introduction of artificial amino acids greatly expands the versatility of the protein sequence pool.…”

Section: Introductionmentioning

confidence: 99%

Incorporation of second coordination sphere d-amino acids alters Cd(II) geometries in designed thiolate-rich proteins

et al. 2017

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We use a de Novo protein design strategy to demonstrate that the second coordination sphere of a metal site plays a key role in controlling coordination geometries of Cd(II)-tris-thiolate complexes. Specifically, we show that alteration of chirality within the core hydrophobic packing region of a three-stranded coiled coil (3SCC) can control the coordination number of Cd(II) by limiting steric encumbrance to the metal center. Within a specific class of 3SCCs [Ac-G-(LKALEEK)n-G-NH2], where n = 4 is TRI and n = 5 is GRAND, one l-Leu may be substituted by l-Cys to generate a planar tris-thiolate array capable of metal binding. In the native peptide containing only the l-configuration of leucine, the three-Cys ligand site leads to a mixture of 3- and 4-coordinate Cd(II). When the l-Leu above (toward the N-terminus) the tris-Cys site is substituted with d-Leu, solely a 3-coordinate structure [Cd(II)S3] was obtained. When d-Leu is located below (toward the C-terminus), a mixture of two coordination geometries, presumably Cd(II)S3O and Cd(II)S3O2, is observed, while substitution with d-Leu both above and below the tris-Cys plane yields a higher percentage of 4-coordinate Cd(II)S3O species. Thus, the use of d-amino acids around a metal’s coordination sphere provides a powerful tool for controlling the properties of future designed metalloproteins.

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Section: Introductionmentioning

confidence: 99%

Incorporation of second coordination sphere d-amino acids alters Cd(II) geometries in designed thiolate-rich proteins

et al. 2017

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“…It gives a measure of mitochondrial complex IV activity, which is determined by the oxidation of cytochrome c from the flow of electrons through cytochrome oxidase (Gianni et al, 2004). Cytochrome c oxidase catalyzes the oxidation of cytochrome c and the reduction of oxygen (Hu and Wang 2016). These reactions are closely tied to energy production and interference with cytochrome c oxidase activity leads to build up of ROS (Ogbi et al, 2004).…”

Section: Cytochrome C Oxidasementioning

confidence: 99%

Effects of decreased dietary vitamin E plus a proprietary antioxidant blend on mitochondria in young performance horses

Owen

White

Brennan

2019

Journal of Equine Veterinary Science

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Mitochondrial health is pertinent to optimizing athletic performance and is maintained in part by dietary antioxidants such as selenium (Se) and vitamin E (vitE). Mitochondrial adaptations to elevated dietary Se coupled with decreased vitE have not yet been determined.Young Quarter Horses (mean ± SEM; 17.6 ± 0.2 mo) were used to test the hypothesis that horses receiving a proprietary antioxidant blend containing Se yeast (EconomasE, Alltech, Inc., Nicholasville, KY) would have improved mitochondrial characteristics compared to horses receiving Se and vitE at current requirements, regardless of reduced vitE intake. Horses were balanced by age, sex, BW, and farm of origin and randomly assigned to one of three customformulated concentrates fed at 1% BW (DM basis): 1) 100 IU vitE/kg DM and 0.1 mg Se/kg DM (CON, n = 6), 2) no added vitE plus EconomasE to provide 0.1 mg Se/kg DM (ESe1, n = 6), or 3) no added vitE plus EconomasE to provide 0.3 mg Se/kg DM (ESe3, n=6). Tissue was collected from the gluteus medius at wk 0 and 12 of dietary treatment and evaluated for mitochondrial enzyme activities by kinetic, colorimetry and mitochondrial capacities by highresolution respirometry. Data were analyzed using PROC MIXED in SAS v9.4 with repeated measures (time) and fixed effects of time, diet, and time × diet; horse(diet) served as a random effect. Mitochondrial number (citrate synthase activity; CS), function (cytochrome c oxidase activity; CCO), and integrated (per mg tissue) oxidative (P) and electron transport (E) capacities increased from wk 0 to 12 in all horses (P ≤ 0.05). Intrinsic (relative to CS) CCO activity and P and E capacities, decreased from wk 0 to 12 (P ≤ 0.02). Horses in CON had higher integrated P with complex I and II substrates (PCI+II), ECI+II, and ECII than ESe1 throughout the study (P ≤ 0.03); integrated ECII was also higher in CON than ESe3 (P = 0.03). Results from the current vi CONTRIBUTORS AND FUNDING SOURCES This work was supervised by a thesis committee consisting of Dr. Sarah White of the

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“…To date, several metal-binding unnatural amino acids (UAAs) have been incorporated site-specifically into proteins in Escherichia coli, including (2,2′-bipyridin-5-yl) alanine (Bpy-Ala), , 2-amino-3-[4-hydroxy-3-(1 H -pyrazol-1-yl) phenyl] propanoic acid (pyTyr), and 2-amino-3-(8-hydroxyquinolin-5-yl) propanoic acid (HQ-Ala). , Mills et al have used a computational approach to design a metal-binding domain in a protein with a very high affinity to Zn 2+ ions ( K d = 40 pM), using the metal-binding UAA Bpy-Ala . In addition, previous results suggested that high metal affinity of these UAAs can function independently to coordinate metal ions without additional coordinating amino acid residues in the metal-binding site . These studies prove that protein engineering by genetic code expansion has advanced into a powerful approach to design metal-binding domains in enzymes in a manner that can change their catalytic activity.…”

Section: Introductionmentioning

confidence: 99%

“…5,4 Mills et al have used a computational approach to design a metal-binding domain in a protein with a very high affinity to Zn 2+ ions (K d = 40 pM), using the metal-binding UAA Bpy-Ala. 19 In addition, previous results suggested that high metal affinity of these UAAs can function independently to coordinate metal ions without additional coordinating amino acid residues in the metalbinding site. 20 These studies prove that protein engineering by genetic code expansion has advanced into a powerful approach to design metal-binding domains in enzymes in a manner that can change their catalytic activity.…”

Section: ■ Introductionmentioning

confidence: 99%

Genetically Expanded Reactive-Oxygen-Tolerant Alcohol Dehydrogenase II

et al. 2020

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Herein, we report the site-specific incorporation of L-3,4-dihydroxyphenylalanine as a promising method to engineer an oxygen-tolerant alcohol dehydrogenase II. The engineered mutant alcohol dehydrogenase II binds Zn 2+ with high binding affinity and is functional under aerobic and oxidative conditions for a longer time than the wild-type, Fe 2+ -binding alcohol dehydrogenase II. Overall, the mutant enzyme demonstrated electrochemical activity toward both acetaldehyde reduction and ethanol oxidation reactions. This enzyme could have a potential use in efficient biofuel production under aerobic conditions in photosynthetic organisms despite the inherent oxygen evolution reaction by photosystem II.

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Method for Enzyme Design with Genetically Encoded Unnatural Amino Acids

Cited by 6 publications

References 55 publications

Incorporation of second coordination sphere d-amino acids alters Cd(II) geometries in designed thiolate-rich proteins

Incorporation of second coordination sphere d-amino acids alters Cd(II) geometries in designed thiolate-rich proteins

Effects of decreased dietary vitamin E plus a proprietary antioxidant blend on mitochondria in young performance horses

Genetically Expanded Reactive-Oxygen-Tolerant Alcohol Dehydrogenase II

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