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  • 1
    Electronic Resource
    Electronic Resource
    Cu(II)-Binding properties of a cytochrome c with a synthetic metal-binding site: His-X3-His in an α-helix (1991)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 10 (1991), S. 156-161 
    Details
    ISSN: 0887-3585
    Keywords: synthetic metalloproteins ; protein engineering ; iso-1-cytochrome c ; metal binding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A metal-binding site consisting of two histidines positioned His-X3-His in an α-helix has been engineered into the surface of Saccharomyces cerevisiae iso-1-cytochrome c. The synthetic metal-binding cytochrome c retains its biological activity in vivo. Its ability to bind chelated Cu(II) has been characterized by partitioning in aqueous two-phase polymer systems containing a polymer-metal complex, Cu(II)IDA-PEG, and by metal-affinity chromatography. The stability constant for the complex formed between Cu(II)IDA-PEG and the cytochrome c His-X3-His site is 5.3 × 104 M-1, which corresponds to a chelate effect that contributes 1.5 kcal mol-1 to the binding energy. Incorporation of the His-X3-His site yields a synthetic metal-binding protein whose metal affinity is sensitive to environmental conditions that alter helix structure or flexibility.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/prot.340100209
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  • 2
    Electronic Resource
    Electronic Resource
    Stabilization of substilisin E in organic solvents by site-directed mutagenesis (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 39 (1992), S. 141-147 
    Details
    ISSN: 0006-3592
    Keywords: protein engineering ; enzymes in organic solvents ; protein stabilization ; subtilisin E ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Subtilisin E was rationally engineered to improve its stability in polar organic solvents such as dimethylformamide (DMF). A charged surface residue, Asp248, was substituted by three amino acids of increasing hydrophobicity, Asn, Ala, and Leu; all three variants were stabilized with respect to wild type in 80% DMF. This stabilization was only observed in the presence of high concentrations of the organic solvent: no stability enhancements were observed in 40% DMF. In contrast, the mutation Asn218 → Ser alters internal hydrogen bonding interactions and stabilizes subtilisin E in both 40% and 80% DMF. This study provides additional evidence that substitution of surface-charged residues is a generally useful mechanism for stabilizing enzymes in organic media and that the stabilizing effects of such substitutions are unique to highly altered solvent environments. The effects of the single amino acid substitutions on free energies of stabilization are additive in the Asp248 → Asn + Asn218 → Ser combination variant, yielding an enzyme that is 3.4 times more stable than wild type in 80% DMF.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260390204
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  • 3
    Electronic Resource
    Electronic Resource
    Random mutagenesis to enhance the activity of subtilisin in organic solvents: Characterization of Q103R subtilisin E (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 39 (1992), S. 658-662 
    Details
    ISSN: 0006-3592
    Keywords: random mutagenesis ; subtilisin ; organic solvents ; enzyme kinetics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Q103R subtilisin E was isolated following random mutagenesis and screening for improved activity in the presence of dimethylformamide (DMF). Our goal is to identify the mechanism(s) by which amino acid substitutions can enhance enzyme activity in polar organic solvents. A quantitative framework for comparing substrate binding and catalytic activities of mutant and wild-type enzymes in the presence and absence of DMF is outlined. Kinetic experiments performed at high salt concentration (1M KCl) reveal that the mechanism behind the Q103R variant's enhanced activity toward succinyl-Ala-Ala-Pro-Phe-p-nitroanilide is both electrostatic and nonelectrostatic in origin. Favorable electrostatic interactions between the negatively charged succinyl group of the substrate and the positive charge on Arg 103 are responsible for tighter substrate binding. This conclusion is supported by kinetic experiments performed on the related substrate Ala-Ala-Pro-Phe-p-nitroanilide and the hydrolysis kinetics of the Q103E, Q103K, and Q103S variants constructed by site-directed mutagenesis. These results highlight the importance of the choice of the substrate used to screen for improvements in catalytic activity.
    Additional Material: 1 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260390610
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  • 4
    Electronic Resource
    Electronic Resource
    A mathematical model for metal affinity protein partitioning (1990)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 35 (1990), S. 682-690 
    Details
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A mathematical model of metal affinity partitioning has been derived and used to describe protein partitioning in Cu (II)PEG/dextran systems. A working model has been extended to account for inhibition, which for metal affinity extraction is the inhibition of protein-metal binding by hydrogen ion. PEG/dextran partitioning experiments were performed on four proteins, tuna heart cytochrome c, Candida krusei cytochrome c, horse myoglobin, and sperm whale myoglobin. The partition coefficients for these proteins are increased by the addition of Cu (II)PEG-IDA, due to the affinity between the chelated copper atom and metal-coordinating histidine residues on the protein surface. The results of experiments to determine the effects of the number of binding sites on the protein, the copper concentration, and pH on partitioning are all well-described by the mathematical model. The pKa value of the metal binding site was determined to be 6.5, which is in the range of pKa values commonly observed for surface histidines. The average association constant for the binding of Cu (II)PEG-IDA to accessible histidines was found to be 4.5 × 103. This value is comparable to stability constants measured by conventional potentiometry techniques for analogous small complexes.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260350705
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  • 5
    Electronic Resource
    Electronic Resource
    Review: Selective ligand-exchange adsorbents prepared by template polymerization (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 48 (1995), S. 431-436 
    Details
    ISSN: 0006-3592
    Keywords: metal-ligand coordination ; molecular recognition ; surface modification ; molecular imprinting ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Highly selective ligand-exchange absorbents have been prepared by template polymerization, a process in which the target molecule serves as a template for assembly of specific recognition sites. In an effort to develop materials suitable for chromatographic separations, thin coatings of the selctive templated polymers have been grafted to two reactive macroporous supports, poly(trimethylolpropane trimethacrylate) (TRIM), and propylmethacrylate-derivatized silica beads. The precursor polymer prepared from the trifunctioal TRIM monomer is macroporous and highly crosslinked, providing a stable structure for surface grafting. The TRIM precursor polymer and various surface-grafted copolymers have been characterized by scanning electron microscopy (SEM) and IR, 13C NMR, and XPS spectroscopic techniques. Composite adsorbents have also been prepared using propylmethacrylate-modified silica particles. While equilibrium rebinding selectivites for both types of surface-templated materials are similar to those reported previously for bulk-polymerized template polymers, the composite materials are far better suited to chromatographic separatios. Highly similar bis-imidazole substrates can be separated by ligand-exchange chromatography on these new templated adsorbents. © 1995 John Wiley & Sons, Inc.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260480504
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  • 6
    Electronic Resource
    Electronic Resource
    Review: Multipoint binding and heterogeneity in immobilized metal affinity chromatography (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 48 (1995), S. 437-443 
    Details
    ISSN: 0006-3592
    Keywords: chromatography ; protein binding ; metal affinity chromatography ; heterogeneous adsorption ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Studies carried out using engineered proteins clearly demonstrate that adsorption to derivatized surfaces involves multiple interactions between functional groups on the protein and complementary sites distributed on the surface. The fact that adsorption involves multipoint interactions has important implications for the design of separations processes and for the interpretation of heterogeneity in biological recognition phenomena. Increasing the density of surface metal sites (immobilized copper ions) is found to be functionally equivalent to increasing the number of metal-coordinating groups on the protein (histidines and deporotonated amines), m in that both processes increase the likelihood of simultaneous interactions between the protein and the surface. A consequence of multiple-site interactions is a significant in crease in protein binding affinity that depends on the arrangement of surface sites. A protein will show the highest affinity for arrangements of surface sites which best match its own pattern of functioal groups and will show lower affinity for less optimal arrangements, resulting in binding that is inherently heterogeneous. We have found that reversible protein adsorption in immobilized metal affinity chromatography (IMAC) is described by the Temikin model, which characterizes binding heterogeneity by a uniform distribution of binding energies over the population of surface binding sites. © 1995 John Wiley & Sons, Inc.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260480505
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