Stability of oxidized Escherichia coli thioredoxin and its dependence on protonation of the aspartic acid residue in the 26 position

Ladbury, John E.; Wynn, Richard; Hellinga, Homme W.; Sturtevant, Julian M.

doi:10.1021/bi00080a026

Cited by 37 publications

(59 citation statements)

References 35 publications

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“…This tendency of the protein to dimerize was observed in the case of the wild-type protein (Ladbury et al, 1993(Ladbury et al, , 1994 and in the L78K and L78R mutants (Ladbury et al, 1995) previously studied. Analytical ultracentrifugation studies suggest dimerization of the wild-type protein (Laurent et al, 1964 …”

Section: Analysis Of Dsc Data Suggests That the Variants Have Differesupporting

confidence: 56%

“…We have selected 120 p M as a standard concentration and the apparent t112 of the L78C* mutant as the temperature for the comparison. This value was obtained from the linear relationship between l/TIl* (T = t1/2 + 273.15) and the natural logarithm of concentration as previously described (Ladbury, 1993;Tanaka, 1993) and AH, , , ( A H at Tl/2) from the plot of AHca, versus concentration. The free energy of unfolding was calculated as described elsewhere (Ladbury et al, 1993).…”

Section: The Thermodynamics Of Unfolding Of the Variant Formsmentioning

confidence: 99%

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The adaptability of Escherichia coli thioredoxin to non‐conservative amino acid substitutions

et al. 1997

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The adaptability of Escherichia coli thioredoxin to the substitution of a series of non-natural amino acids has been investigated. Different thiosulfonated alkyl groups were inserted into the hydrophobic core of the protein in position 78 via disulfide bonding with a buried cysteine residue as previously described (Wynn R, Richards F M . 1993. Unnatural amino acid packing mutants of Escherichia coli thioredoxin produced by combined mutagenesis/chemical modification techniques. Protein Sci 2:395-403). The side chains added to the cysteine included methyl, ethyl, n-propyl, n-butyl, n-pentyl, and cyclo-pentyl derivatives. The side chains appear to exploit the presence of the large cavities to incorporate these variant forms, enabling the protein to fold and have some activity. Solution structural and kinetic data suggested that these substitutions had little effect on the overall fold of the protein. Thermodynamic data revealed that the entropic effect of restricting the side chains in the folded protein has an effect on the stability. The variant forms of thioredoxin have different propensities to form dimers despite the limited structural perturbations. Molecular modeling studies allow the conformation of the side chains to be assessed.

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Section: Analysis Of Dsc Data Suggests That the Variants Have Differesupporting

confidence: 56%

Section: The Thermodynamics Of Unfolding Of the Variant Formsmentioning

confidence: 99%

The adaptability of Escherichia coli thioredoxin to non‐conservative amino acid substitutions

et al. 1997

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“…The well studied protein Escherichia coli thioredoxin (33) was selected as a scaffold because of its favorable expression properties, thermodynamic stability (34), and successful history in computational design (15). Naturally occurring thioredoxin can supply the reducing power for ribonucleotide reductase by way of its disulfide bond, but is essentially catalytically inert with respect to PNPA binding and hydrolysis.…”

Section: Resultsmentioning

confidence: 99%

Enzyme-like proteins by computational design

Bolon

Mayo

2001

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

367

299

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We report the development and initial experimental validation of a computational design procedure aimed at generating enzymelike protein catalysts called ''protozymes.'' Our design approach utilizes a ''compute and build'' strategy that is based on the physical͞chemical principles governing protein stability and catalytic mechanism. By using the catalytically inert 108-residue Escherichia coli thioredoxin as a scaffold, the histidine-mediated nucleophilic hydrolysis of p-nitrophenyl acetate as a model reaction, and the ORBIT protein design software to compute sequences, an active site scan identified two promising catalytic positions and surrounding active-site mutations required for substrate binding. Experimentally, both candidate protozymes demonstrated catalytic activity significantly above background. One of the proteins, PZD2, displayed ''burst'' phase kinetics at high substrate concentrations, consistent with the formation of a stable enzyme intermediate. The kinetic parameters of PZD2 are comparable to early catalytic Abs. But, unlike catalytic Ab design, our design procedure is independent of fold, suggesting a possible mechanism for examining the relationships between protein fold and the evolvability of protein function.A prominent goal of protein design is the generation of proteins with novel functions, including the catalytic rate enhancement of chemical reactions. The ability to design an enzyme to perform a given chemical reaction has considerable practical application for industry and medicine, particularly for the synthesis of pharmaceuticals (1). Significant progress has been made at enhancing the catalytic properties of existing enzymes through directed evolution (2). In contrast, the design of proteins with novel catalytic properties has met with relatively limited success (3-5). We present a general computational approach for the design of enzyme-like proteins with novel catalytic activities.The use of transition-state analogs as haptens to elicit catalytic Abs has been the most successful technique to date for generating novel protein catalysts (6). Natural enzymes combine transition-state stabilization with precisely oriented catalytic side chains. Although a reactive hapten has been used in the generation of an Ab with a powerful nucleophile at the active site (7), current catalytic Ab technology does not efficiently select for both catalytic side chains and tight noncovalent affinity in the same molecule. The relationship between the general backbone fold of an enzyme and its catalytic properties is not well understood. This observation is particularly relevant to catalytic Abs that are currently constrained to the Ab fold and which have yet to show catalytic activity on par with natural enzymes.Rational design efforts have recently succeeded at altering the catalytic reactions of two different enzymes (8, 9). Cyclophilin, a cis-trans isomerase of X-Pro peptide bonds, was engineered into an endopeptidase by grafting a triad of catalytic residues commonly found in serine proteases at the...

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“…°C for the reduced form (12,13). When thioredoxin is fused to other proteins, it can improve their solubility and, especially when in the oxidized form, improve their thermal stability, allowing a heat step during purification.…”

Section: Resultsmentioning

confidence: 99%

Human RAD52 Protein Has Extreme Thermal Stability

et al. 2001

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The human RAD52 protein plays an important role in the earliest stages of chromosomal double-strand break repair via the homologous recombination pathway. Individual subunits of RAD52 associate into seven-membered rings. These rings can form higher order complexes. RAD52 binds to DNA breaks, and recent studies suggest that the higher order self-association of the rings promotes DNA end joining. Monomers of the RAD52(1-192) deletion mutant also associate into ring structures but do not form higher order complexes. The thermal stability of wild-type and mutant RAD52 was studied by differential scanning calorimetry. Three thermal transitions (labeled A, B, and C) were observed with melting temperatures of 38.8, 73.1, and 115.2°C. The RAD52(1-192) mutant had only two thermal transitions at 47.6 and 100.9°C (labeled B and C). Transitions were labeled such that transition C corresponds to complete unfolding of the protein. The effect of temperature and protein concentration on RAD52 self-association was analyzed by dynamic light scattering. From these data a four-state hypothetical model was developed to explain the thermal denaturation profile of wild-type RAD52. The three thermal transitions in this model were assigned as follows. Transition A was attributed to the disruption of higher order assemblies of RAD52 rings, transition B to the disruption of rings to individual subunits, and transition C to complete unfolding. The ring-shaped quaternary structure of RAD52 and the formation of higher ordered complexes of rings appear to contribute to the extreme stability of RAD52. Higher ordered complexes of rings are stable at physiological temperatures in vitro.RAD52 1 protein plays a critical role in mitotic and meiotic recombination as well as double-strand break repair (1, 2). On the basis of a series of protein-protein interaction assays and DNA binding studies (3-5), a domain map of human RAD52 (RAD52) was proposed by Park et al. (Figure 1). Electron microscopy (EM) studies of Saccharomyces cereVisiae and human RAD52 have revealed formation of ringshaped structures (9-13 nm in diameter), as well as higher order aggregates (6-8). The RAD52 rings appear to be composed of seven subunits (9). EM studies also showed that RAD52 recognizes and binds to double-stranded DNA ends as an aggregated complex that ranges in size from approximately 15 to 60 nm in diameter (8). This binding promoted end-to-end association between DNA molecules and stimulated the ligation of both cohesive and blunt DNA ends (8). Recently, by studying wild type and two deletion mutants of RAD52 (Figure 1), we demonstrated that the selfassociation domain in the N-terminal half of RAD52 is responsible for ring formation and that elements in the C-terminal half of the molecule participate in the formation of higher order complexes of rings (10).Due to the biological interest of human RAD52 and the apparent biochemical importance of RAD52 self-association in DNA repair, we studied its multiple levels of selfassociation and stability using biophy...

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Stability of oxidized Escherichia coli thioredoxin and its dependence on protonation of the aspartic acid residue in the 26 position

Cited by 37 publications

References 35 publications

The adaptability of Escherichia coli thioredoxin to non‐conservative amino acid substitutions

The adaptability of Escherichia coli thioredoxin to non‐conservative amino acid substitutions

Enzyme-like proteins by computational design

Human RAD52 Protein Has Extreme Thermal Stability

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