Unnatural amino acid packing mutants of <i>Escherichia coli</i> thioredoxin produced by combined mutagenesis/chemical modification techniques

Wynn, Richard; Richards, Frederic M.

doi:10.1002/pro.5560020311

Cited by 42 publications

(37 citation statements)

References 60 publications

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“…Trx is a stable protein (oxidized Trx T m , 86ЊC; reduced Trx T m , 75ЊC) (15) that accommodates conservative as well as some nonconservative mutations (18). In addition to the availability of a high-resolution (1.68 Å) x-ray structure (19), there are several solution structures (oxidized and reduced) derived from NMR spectroscopic methods (20,21), indicating the feasibility of obtaining structural information on the designed protein in the absence of crystals.…”

Section: Resultsmentioning

confidence: 99%

Construction of a catalytically active iron superoxide dismutase by rational protein design

Pinto

Hellinga

Caradonna

1997

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

121

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The rational protein design algorithm DEZYMER was used to introduce the active site of nonheme iron superoxide dismutase (SOD) into the hydrophobic interior of the host protein, Escherichia coli thioredoxin (Trx), a protein that does not naturally contain a transition metalbinding site. Reconstitution of the designed protein, Trx-SOD, showed the incorporation of one high-affinity metal-binding site. The electronic spectra of the holoprotein and its N 3 ؊ and F ؊ adducts are analogous to those previously reported for native {Fe 3؉ }SOD. Activity assays showed that {Fe 3؉ }Trx-SOD is capable of catalyzing the dismutation of the superoxide anion; comparative studies with the unrelated wild-type E. coli iron SOD indicated that {Fe 3؉ }Trx-SOD catalyzes the dismutation reaction at a rate on the order of 10 5 M ؊1 s ؊1 . The ability to design catalytically competent metalloenzymes allows for the systematic investigation of fundamental mechanistic questions concerning catalysis at transition metal centers.Protein design methodologies (1-3) have recently focused on incorporating transition metal ions into proteins (3-6) to take advantage of their well-documented catalytic or electron transfer functions. However, this long-term objective of rationally engineering metalloproteins to introduce novel catalytic functions has heretofore met with only limited success (5-8). We have used the rational protein design algorithm DEZYMER (9) to test our ability to design catalytically active metalloenzymes by constructing a five-coordinate nonheme iron active site analogous to that found in iron-dependent superoxide dismutase (SOD) (10, 11) in a host protein, thioredoxin (Trx) (Fig. 1). The construction of this site represents a significantly different challenge from the design of coordinatively saturated, enzymatically inert, metal sites (6,8,40) by attempting to build a site that catalyzes the inner sphere transfer of electrons both to and from small molecule substrates. For this chemistry to occur, the availability of an open coordination position, or one occupied by a labile ligand such as H 2 O, is required, but the site must be sufficiently buried to prevent protein dimerization via a metal bridge. Introduction of a cavity for anion binding, as well as other factors relating to the attraction and transport of the anion to the active site, need to be considered as well.The DEZYMER algorithm makes predictions based on strict geometric principles without explicit consideration of binding thermodynamics or protein dynamics. The rational design approach used here is based on the placement of an active site into the framework of a known protein fold. Active sites are described as geometrical arrangements of functionally important amino acids around a ligand, in this case a transition metal center. In the first phase of the search, the DEZYMER algorithm systematically examines a protein structure to identify backbone positions that are arranged in such a way that appropriate rotamers of the residues in the binding-site defini...

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Section: Resultsmentioning

confidence: 99%

Construction of a catalytically active iron superoxide dismutase by rational protein design

Pinto

Hellinga

Caradonna

1997

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

121

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“…Trx is a stable protein (oxidized Trx T m , 86°C; reduced Trx T m , 75°C) (30) that accommodates conservative as well as some nonconservative mutations (32). In addition to the availability of a highresolution (1.68 Å) x-ray structure (33), there are several solution structures (oxidized and reduced) derived from NMR spectroscopic methods (34,35), indicating the feasibility of obtaining structural information on the designed protein in the absence of crystals.…”

Section: Rational Design the [Fe 4 S 4 Cys 4 ]mentioning

confidence: 99%

The rational design and construction of a cuboidal iron–sulfur protein

Coldren

Hellinga²,

Caradonna³

1997

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

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Metalloproteins offer a particularly interesting target for the design of function, because the biological chemistry of metals is extraordinarily rich. Cuboidal [Fe 4 S 4 ] clusters are among the most common electron-transfer centers found in biology (1). These clusters act as either simple soluble electron-transfer agents, membrane-bound components of electron-transfer chains, or parts of the electron reservoir found in complex metalloenzymes in plants, animals, and bacteria. In addition to being intimately involved in electron-transport systems and in the metabolism of carbon, oxygen, hydrogen, sulfur, and nitrogen, studies have suggested that these centers also exhibit gene regulatory, catalytic, and structural functions, and have been found as part of a morphogenetic protein (1).The successful construction of a functional metal center requires that three sets of factors are taken into account: (i) the construction of a correctly folded protein, (ii) the coordination requirements of the metal, (iii) the modulation of the properties of the metal center and protein matrix to achieve the required control of reactivity. Thus far, metalloprotein designs have focused primarily on the first two factors (2-8). The redox properties of [Fe 4 S 4 ] centers, which are strongly dependent on the protein environment, offer a good system to explore the modulation of the metal center by the protein (1, 9). Cuboidal [Fe 4 S 4 ] proteins, which contain structurally equivalent metal clusters, are grouped in two classes, the ferredoxins (Fds) and the high-potential iron-sulfur proteins (HiPIPs couple (E o ϭ ϩ100 mV to ϩ450 mV). Although several high-resolution x-ray crystallographic structures of HiPIP systems as well as 4Fe-, 7Fe-, and 8Fe-Fds are available (1), the identification of those factors that determine the redox couple are just beginning to emerge. Conjectures have been presented that cluster-protein (amide NOH to cluster S 2Ϫ or cysteinyl S) hydrogen bonding (10), solvent accessibility of cluster, and electrostatic field gradients arising from the presence of acidic, basic, and hydrophobic residues in the vicinity of the [Fe 4 S 4 ] cluster, are important in determining the redox potential (9). Spectroscopic studies (11), ab inito calculations (12), and computer simulations (13, 14) support these factors as determinants of cluster redox potential. Studies of model systems (15), mutant iron-sulfur proteins (16,17), and interspecies variants (18) have also indicated the importance of the protein matrix and solvent. In addition, recent quantitative modeling simulations of the redox potentials of iron-sulfur proteins using the protein dipoles Langevin dipoles model have implicated the Coulombic interaction of the cluster with the protein atom charges as a major determinant of the variations in measured redox potentials (9). Specifically, the proximity and orientation of protein matrix dipoles arising from the parallel alignment of trans amide NOH and CAO bonds, which depends on the intrinsic structure of the polyp...

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“…E. coli thioredoxin is a particularly difficult molecule to crystallize, and this has inhibited structural characterization of the many mutants of this protein that have been constructed. [40][41][42][43] The activities of these mutant dimers were examined. In addition, a database analysis was carried out to examine the general applicability of this methodology to other proteins.…”

Section: Introductionmentioning

confidence: 99%

Design of Disulfide-linked Thioredoxin Dimers and Multimers Through Analysis of Crystal Contacts

Das

Kobayashi

Yamada

et al. 2007

Journal of Molecular Biology

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Unnatural amino acid packing mutants of Escherichia coli thioredoxin produced by combined mutagenesis/chemical modification techniques

Cited by 42 publications

References 60 publications

Construction of a catalytically active iron superoxide dismutase by rational protein design

Construction of a catalytically active iron superoxide dismutase by rational protein design

The rational design and construction of a cuboidal iron–sulfur protein

Design of Disulfide-linked Thioredoxin Dimers and Multimers Through Analysis of Crystal Contacts

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