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At least 703 records · Page 39Linked to original sources
PubMed2000

Single-step purification of a recombinant thermostable alpha-amylase after solubilization of the enzyme from insoluble aggregates.

The expression of the gene encoding a thermostable alpha-amylase (EC 3.2.1.1) (optimal activity at 100 degrees C) from the hyperthermophilic archaeon Pyrococcus woesei in the mesophilic hosts Escherichia coli and Halomonas elongata resulted in the formation of insoluble aggregates. More than 85% of the recombinant enzyme was present within the cells as insoluble but catalytically active aggregates. The recombinant alpha-amylase was purified to homogeneity in a single step by hydrophobic interaction chromatography on a phenyl superose column after solubilization of the enzyme under nondenaturing conditions. The enzyme was purified 258-fold with a final yield of 54%.

Chromatography, Liquid↗
PubMed2003

Nucleotide-induced conformational changes in an isolated Escherichia coli DNA polymerase III clamp loader subunit.

Sliding clamps are loaded onto DNA by ATP-driven clamp loader complexes. The structure of the E. coli clamp loader in a nucleotide-free state has been determined previously. We now report crystal structures of a truncated form of the isolated gamma-ATPase subunit, gamma(1-243), of the E. coli clamp loader, in nucleotide-free and bound forms. The gamma subunit adopts a defined conformation when empty, in which the nucleotide binding site is blocked. The binding of either ATPgammaS or ADP, which are shown to bind with equal affinity to gamma(1-243), induces a change in the relative orientation of the two domains such that nucleotides can be accommodated. This change would break one of the gamma:gamma interfaces seen in the empty clamp loader complex, and may represent one step in the activation process.

Adenosine Triphosphatases↗
PubMed2006

Geometries and electronic structures of cyanide adducts of the non-heme iron active site of superoxide reductases: vibrational and ENDOR studies.

We have added cyanide to oxidized 1Fe and 2Fe superoxide reductase (SOR) as a surrogate for the putative ferric-(hydro)peroxo intermediate in the reaction of the enzymes with superoxide and have used vibrational and ENDOR spectroscopies to study the properties of the active site paramagnetic iron center. Addition of cyanide changes the active site iron center in oxidized SOR from rhombic high-spin ferric (S = 5/2) to axial-like low-spin ferric (S = 1/2). Low-temperature resonance Raman and ENDOR data show that the bound cyanide adopts three distinct conformations in Fe(III)-CN SOR. On the basis of 13CN, C15N, and 13C15N isotope shifts of the Fe-CN stretching/Fe-C-N bending modes, resonance Raman studies of 1Fe-SOR indicate one near-linear conformation (Fe-C-N angle approximately 175 degrees) and two distinct bent conformations (Fe-C-N angles <140 degrees). FTIR studies of 1Fe-SOR at ambient temperatures reveals three bound C-N stretching frequencies in the oxidized (ferric) state and one in the reduced (ferrous) state, indicating that the conformational heterogeneity in cyanide binding is a characteristic of the ferric state and is not caused by freezing-in of conformational substates at low temperature. 13C-ENDOR spectra for the 13CN-bound ferric active sites in both 1Fe- and 2Fe-SORs also show three well-resolved Fe-C-N conformations. Analysis of the 13C hyperfine tensors for the three substates of the 2Fe-SOR within a simple heuristic model for the Fe-C bonding gives values for the Fe-C-N angles in the three substates of ca. 123 degrees (C3) and 133 degrees (C2), taking a reference value from vibrational studies of 175 degrees (C1 species). Resonance Raman and ENDOR studies of SOR variants, in which the conserved glutamate and lysine residues in a flexible loop above the substrate binding pocket have been individually replaced by alanine, indicate that the side chains of these two residues are not involved in direct interaction with bound cyanide. The implications of these results for understanding the mechanism of SOR are discussed.

Binding Sites↗
PubMed1999

NMR observation of selected segments in a larger protein: central-segment isotope labeling through intein-mediated ligation.

Peptide segments in a protein, which can include an active site of interest or be a series of parts constituting the entire structure, are now selectively observed by nuclear magnetic resonance (NMR) spectroscopy using samples prepared by the intein-mediated ligation method. Two separate inteins were used to ligate NMR-transparent segments to both the ends of an NMR-visible segment, producing a partly visible intact protein molecule. The (15)N-(1)H correlation spectrum of a 370-residue maltose binding protein labeled with (15)N at a continuous segment comprising residues Gly(101)-Ser(238) showed the essential elimination of signal overlapping, the signals being at the same positions as for the uniformly labeled sample. This method will allow structural analysis by NMR of over 50-kDa proteins in combination with contemporary NMR techniques suppressing the signal decays of larger proteins.

ATP-Binding Cassette Transporters↗
PubMed2000

Contribution of surface salt bridges to protein stability.

The role of surface salt bridges in protein stabilization has been a source of controversy. Here we present the NMR structure of a hyperthermophilic rubredoxin variant (PFRD-XC4) and the thermodynamic analysis of two surface salt bridges by double mutant cycles. This analysis shows that the surface side chain to side chain salt bridge between Lys 6 and Glu 49 does not stabilize PFRD-XC4. The main chain to side chain salt bridge between the N-terminus and Glu 14 was, however, found to stabilize PFRD-XC4 by 1. 5 kcal mol(-)(1). The entropic cost of making a surface salt bridge involving the protein's backbone is reduced, since the backbone has already been immobilized upon protein folding.

Bacterial Proteins↗
PubMed2002

Sterically hindered carboxylate ligands support water-bridged dimetallic centers that model features of metallohydrolase active sites.

The synthesis and characterization of carboxylate-bridged dimetallic complexes are described. By using m-terphenyl-derived carboxylate ligands, a series of dicobalt(II), dicobalt(III), dinickel(II), and dizinc(II) complexes were synthesized. The compounds are [Co(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)] (1), [Co(2)(mu-OH(2))(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)] (2a-c), [Co(2)(mu-OH)(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)] (3), [Ni(2)(mu-O(2)CAr(Tol))(4)L(2)] (4), [Ni(2)(mu-HO...H)(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)] (5), and [Zn(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)] (6), where Ar(Tol)CO(2)H = 2,6-di(p-tolyl)benzoic acid and L = pyridine, THF, or N,N-dibenzylethylenediamine. Structural analysis of these complexes revealed that additional bridging ligands can be readily accommodated within the [M(2)(mu-O(2)CAr(Tol))(2)](2+) core, allowing a wide distribution of M...M distances from 2.5745(6) to 4.0169(9) A. Unprecedented bridging units [M(2)(mu-OH(2))(2)(mu-O(2)CR)(2)](n+) and [M(2)(mu-HO...H)(2)(mu-O(2)CR)(2)](n+) were identified in 2a-c and 5, respectively, in which strong hydrogen bonding accommodates shifts of protons from bridging water molecules toward the dangling oxygen atoms of terminal monodentate carboxylate groups. Such a proton shift along the O...H...O coordinate attenuates the donor ability of the anionic carboxylate ligand, which can translate into increased Lewis acidity at the metal centers. Such double activation of bridging water molecules by a Lewis acidic metal center and a metal-bound general base may facilitate the reactivity of metallohydrolases such as methionine aminopeptidase (MAP).

Aminopeptidases↗
PubMed2003

Computational study of the non-heme iron active site in superoxide reductase and its reaction with superoxide.

The ferrous square-pyramidal [Fe(NHis)4(SCys)] site of superoxide reductases (SORs) has been shown to reduce superoxide at a nearly diffusion-controlled rate. The final products of the reaction are hydrogen peroxide and the ferric hexacoordinated SOR site, with a carboxylate group from a conserved glutamate serving as the sixth ligand trans to the cysteine sulfur. A transient intermediate absorbing at approximately 600 nm in the reaction of the ferrous pentacoordinated site with superoxide has been proposed to be a ferric-(hydro)peroxo complex (Coulter, E.; Emerson, J.; Kurtz, D. M., Jr.; Cabelli, D. J. Am. Chem. Soc. 2000, 122, 11555-11556.). In the present study, DFT and ZINDO/S-CI results are shown to support the description of the 600-nm intermediate as an end-on, low-spin ferricperoxo or--hydroperoxo complex. Side-on peroxo coordination was found to be significantly less stable than end-on because of constraints on the imidazole ligand ring orientations imposed mostly by the protein. The modeled ferric-hydroperoxo complex had a decidedly nonplanar CysC beta-S-Fe-O-O geometry that appears to be imposed by the same constraints. A single prominent visible absorption of the (hydro)peroxo model is shown to be due mainly to a CysS-->Fe(III) pi charge transfer (CT) transition with a minor portion of His-->Fe(III) pi CT character and very little peroxo-->Fe(III) CT character. On the basis of calculations of models with various mono- and diprotonated peroxo ligands, protonation of the iron-bound peroxo oxygen is a key step in the decay of the ferric(hydro)peroxo complex favoring release of hydrogen peroxide over cleavage of the O-O bond, as occurs in the heme structural analogue, cytochrome P450.

Binding Sites↗
PubMed2001

A dipolar coupling based strategy for simultaneous resonance assignment and structure determination of protein backbones.

A new approach for simultaneous protein backbone resonance assignment and structure determination by NMR is introduced. This approach relies on recent advances in high-resolution NMR spectroscopy that allow observation of anisotropic interactions, such as dipolar couplings, from proteins partially aligned in field ordered media. Residual dipolar couplings are used for both geometric information and a filter in the assembly of residues in a sequential manner. Experimental data were collected in less than one week on a small redox protein, rubredoxin, that was 15N enriched but not enriched above 1% natural abundance in 13C. Given the acceleration possible with partial 13C enrichment, the protocol described should provide a very rapid route to protein structure determination. This is critical for the structural genomics initiative where protein expression and structural determination in a high-throughput manner will be needed.

Models, Molecular↗
PubMed2003

Observation of Fe-H/D modes by nuclear resonant vibrational spectroscopy.

Metal-hydrogen bonding is important in chemistry and catalysis, but H atoms are often difficult to observe, especially in metalloproteins. In this work we show that Fe-H interactions can be probed by nuclear resonance vibrational spectroscopy at the 14.4 keV 57Fe nuclear resonance. An important advantage of this method, compared to Raman and IR spectroscopy, is the selectivity for modes that involve 57Fe motion. We present data on the FeS4 site in rubredoxin and the [FeH(D)6]2- ion. Prospects for studying more complex systems are discussed.

Hydrogen↗
PubMed2005

De novo design of a redox-active minimal rubredoxin mimic.

Metal-binding sites in metalloproteins frequently occur at the interfaces of elements of secondary structure, which has enabled the retrostructural analysis of natural proteins and the de novo design of helical bundles that bind metal ion cofactors. However, the design of metalloproteins containing beta-structure is less well developed, despite the frequent occurrence of beta-conformations in natural metalloproteins. Here, we describe the design and construction of a beta-protein, RM1, that forms a stable, redox-active 4-Cys thiolate Fe(II/III) site analogous to the active site of rubredoxin. The protein folds into a beta-structure in the presence and absence of metal ions and binds Fe(II/III) to form a redox-active site that is stable to repeated cycles of oxidation and reduction, even in an aerobic environment.

Binding Sites↗
PubMed2004

Discovery of the chemical function of glycosidases: design, synthesis, and evaluation of mass-differentiated carbohydrate libraries.

[reaction: see text] Discovery of the catalytic chemical function of the many putative glycosidases coded in genomes currently relies on individual testing of possible substrates, usually as their p-nitrophenol conjugate. Herein, we present an alternative chemical proteomics approach using a synthetic mass-differentiated heat-stable substrate library with mass spectrometry readout. Library components do not serve as reaction inhibitors and both primary and secondary enzyme substrates can be delineated.

Carbohydrates↗
PubMed1999

Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes.

Flap endonucleases (FENs) isolated from archaea are shown to recognize and cleave a structure formed when two overlapping oligonucleotides hybridize to a target DNA strand. The downstream oligonucleotide probe is cleaved, and the precise site of cleavage is dependent on the amount of overlap with the upstream oligonucleotide. We have demonstrated that use of thermostable archaeal FENs allows the reaction to be performed at temperatures that promote probe turnover without the need for temperature cycling. The resulting amplification of the cleavage signal enables the detection of specific DNA targets at sub-attomole levels within complex mixtures. Moreover, we provide evidence that this cleavage is sufficiently specific to enable discrimination of single-base differences and can differentiate homozygotes from heterozygotes in single-copy genes in genomic DNA.

Archaeoglobus fulgidus↗