11 results on '"Archer, D."'
Search Results
2. IscA, an alternate scaffold for Fe-S cluster biosynthesis
- Author
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Krebs, Carsten, Agar, Jeffrey N., Smith, Archer D., Frazzon, Jeverson, Dean, Dennis R., Huynh, Boi Hanh, and Johnson, Michael K.
- Subjects
Nitrogen -- Fixation ,Enzymes -- Synthesis ,Microbial metabolism -- Physiological aspects ,Enzyme kinetics -- Analysis ,Biological sciences ,Chemistry - Abstract
Results demonstrate the ability of Isc homologue (sup)Nif IscA of Azotobacter vinelandii to bind iron and assemble iron-sulfur clusters through a NifS-directed pathway. Data indicate that (sup)Nif IscA lends itself as an alternative scaffold to NifU for the NifS-directed sequential assembly of iron-sulfur clusters.
- Published
- 2001
3. An Isc-type extremely thermostable [2Fe-2S] ferredoxin from Aquifex aeolicus. biochemical, spectroscopic, and unforlding studies
- Author
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Mitou, Geraldine, Higgins, Catherine, Wittung-Stafshede, Pernilla, Conover, Richard C., Smith, Archer D., Johnson, Michael K., Gaillard, Jacques, Stubna, Audria, Munck, Eckard, and Meyer, Jacques
- Subjects
Protein folding -- Analysis ,Bacterial proteins -- Analysis ,Enzymes -- Structure-activity relationship ,Biological sciences ,Chemistry - Abstract
A plant- and mammalian-type [2Fe-2S] ferredoxin, called ferredoxin 5 when expressed in Escherichia coli , is thermostable and differs from the iron-sulfur cluster ferredoxins by exhibiting deletions at N- and C-termini. Spectral analysis indicate that ferredoxin 5 and the Isc-Fd share structural similarities and redox potential properties.
- Published
- 2003
4. NifS-Mediated Assembly of [4Fe−4S] Clusters in the N- and C-Terminal Domains of the NifU Scaffold Protein
- Author
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Carsten Krebs, Jeverson Frazzon, Guy N. L. Jameson, Dennis R. Dean, Boi Hanh Huynh, Michael K. Johnson, Archer D. Smith, Sunil Naik, Jeffrey N. Agar, and Patricia C. Dos Santos
- Subjects
Iron-Sulfur Proteins ,Scaffold protein ,Scaffold ,Stereochemistry ,Spectrum Analysis ,Biology ,Biochemistry ,Protein Structure, Tertiary ,Crystallography ,Bacterial Proteins ,Nitrogen Fixation ,Nitrogenase ,Time course ,Cluster (physics) ,Dimerization ,Transaminases - Abstract
NifU is a homodimeric modular protein comprising N- and C-terminal domains and a central domain with a redox-active [2Fe-2S](2+,+) cluster. It plays a crucial role as a scaffold protein for the assembly of the Fe-S clusters required for the maturation of nif-specific Fe-S proteins. In this work, the time course and products of in vitro NifS-mediated iron-sulfur cluster assembly on full-length NifU and truncated forms involving only the N-terminal domain or the central and C-terminal domains have been investigated using UV-vis absorption and Mössbauer spectroscopies, coupled with analytical studies. The results demonstrate sequential assembly of labile [2Fe-2S](2+) and [4Fe-4S](2+) clusters in the U-type N-terminal scaffolding domain and the assembly of [4Fe-4S](2+) clusters in the Nfu-type C-terminal scaffolding domain. Both scaffolding domains of NifU are shown to be competent for in vitro maturation of nitrogenase component proteins, as evidenced by rapid transfer of [4Fe-4S](2+) clusters preassembled on either the N- or C-terminal domains to the apo nitrogenase Fe protein. Mutagenesis studies indicate that a conserved aspartate (Asp37) plays a critical role in mediating cluster transfer. The assembly and transfer of clusters on NifU are compared with results reported for U- and Nfu-type scaffold proteins, and the need for two functional Fe-S cluster scaffolding domains on NifU is discussed.
- Published
- 2005
5. A Conformational Mimic of the MgATP-Bound 'On State' of the Nitrogenase Iron Protein
- Author
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Michael K. Johnson, Archer D. Smith, Robert Y. Igarashi, Sanchayita Sen, Lance C. Seefeldt, and John W. Peters
- Subjects
Iron-Sulfur Proteins ,Models, Molecular ,Molybdoferredoxin ,Conformational change ,Protein Conformation ,Protein subunit ,Crystal structure ,Crystallography, X-Ray ,Spectrum Analysis, Raman ,Biochemistry ,law.invention ,Adenosine Triphosphate ,Protein structure ,Bacterial Proteins ,Leucine ,Oxidoreductase ,law ,Electron paramagnetic resonance ,chemistry.chemical_classification ,Azotobacter vinelandii ,Binding Sites ,Molecular Mimicry ,Nitrogenase ,Resonance (chemistry) ,Crystallography ,chemistry ,Mutagenesis, Site-Directed ,Spectrophotometry, Ultraviolet ,Oxidoreductases ,Protein Binding - Abstract
The crystal structure of a nitrogenase Fe protein single site deletion variant reveals a distinctly new conformation of the Fe protein and indicates that, upon binding of MgATP, the Fe protein undergoes a dramatic conformational change that is largely manifested in the rigid-body reorientation of the homodimeric Fe protein subunits with respect to one another. The observed conformational state allows the rationalization of a model of structurally and chemically complementary interactions that occur upon initial complex formation with the MoFe protein component that are distinct from the protein-protein interactions that have been characterized previously for stabilized nitrogenase complexes. The crystallographic results, in combination with complementary UV-visible absorption, EPR, and resonance Raman spectroscopic data, indicate that the [4Fe-4S] cluster of both the Fe protein deletion variant and the native Fe protein in the presence of MgATP can reversibly cycle between a regular cubane-type [4Fe-4S] cluster in the reduced state and a cleaved form involving two [2Fe-2S] fragments in the oxidized state. Resonance Raman studies indicate that this novel cluster conversion is induced by glycerol, and the crystallographic data suggest that glycerol is bound as a bridging bidentate ligand to both [2Fe-2S] cluster fragments in the oxidized state.
- Published
- 2004
6. An Isc-Type Extremely Thermostable [2Fe−2S] Ferredoxin from Aquifex aeolicus. Biochemical, Spectroscopic, and Unfolding Studies
- Author
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Michael K. Johnson, Archer D. Smith, Jacques Gaillard, Pernilla Wittung-Stafshede, Jacques Meyer, Géraldine Mitou, Audria Stubna, Catherine L. Higgins, Eckard MüNCK, and Richard C. Conover
- Subjects
Iron-Sulfur Proteins ,inorganic chemicals ,Protein Denaturation ,Protein Folding ,Circular dichroism ,Hot Temperature ,Molecular Sequence Data ,Spectrum Analysis, Raman ,medicine.disease_cause ,Biochemistry ,Spectroscopy, Mossbauer ,medicine ,Amino Acid Sequence ,Cloning, Molecular ,Escherichia coli ,Peptide sequence ,Ferredoxin ,Aquifex aeolicus ,Base Sequence ,biology ,Circular Dichroism ,Electron Spin Resonance Spectroscopy ,biology.organism_classification ,Crystallography ,Azotobacter vinelandii ,Ferredoxins ,bacteria ,Spectrophotometry, Ultraviolet ,Protein folding ,Oxidation-Reduction ,Cysteine - Abstract
Analysis of the genome of the hyperthermophilic bacterium Aquifex aeolicus has revealed the presence of a previously undetected gene potentially encoding a plant- and mammalian-type [2Fe-2S] ferredoxin. Expression of that gene in Escherichia coli has yielded a novel thermostable [2Fe-2S] ferredoxin (designated ferredoxin 5) whose sequence is most similar to those of ferredoxins involved in the assembly of iron-sulfur clusters (Isc-Fd). It nevertheless differs from the latter proteins by having deletions near its N- and C-termini, and no cysteine residues other than those involved in [2Fe-2S] cluster coordination. Resonance Raman, low-temperature MCD and EPR studies show close spectral similarities between ferredoxin 5 and the Isc-Fd from Azotobacter vinelandii. Mössbauer spectra of the reduced protein were analyzed with an S = 1/2 spin Hamiltonian and interpreted in the framework of the ligand field model proposed by Bertrand and Gayda. The redox potential of A. aeolicus ferredoxin 5 (-390 mV) is in keeping with its relatedness to Isc-Fd. Unfolding experiments showed that A. aeolicus ferredoxin 5 is highly thermostable (T(m) = 106 degrees C at pH 7), despite being devoid of features (e.g., high content of charged residues) usually associated with extreme thermal stability. Searches for genes potentially encoding plant-type [2Fe-2S] ferredoxins have been performed on the sequenced genomes of hyperthermophilic organisms. None other than the two proteins from A. aeolicus were retrieved, indicating that this otherwise widely distributed group of proteins is barely represented among hyperthermophiles.
- Published
- 2003
7. IscA, an alternate scaffold for Fe-S cluster biosynthesis
- Author
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Michael K. Johnson, Jeverson Frazzon, Jeffrey N. Agar, Archer D. Smith, Boi Hanh Huynh, Dennis R. Dean, and Carsten Krebs
- Subjects
Iron-sulfur cluster assembly ,Scaffold protein ,Iron-Sulfur Proteins ,Stereochemistry ,Molecular Sequence Data ,Biochemistry ,Cofactor ,Catalysis ,Bacterial Proteins ,Iron-Binding Proteins ,Nitrogen Fixation ,Amino Acid Sequence ,Azotobacter vinelandii ,biology ,Cysteine desulfurase ,Chemistry ,biochemical phenomena, metabolism, and nutrition ,Transferrin-Binding Proteins ,biology.organism_classification ,Kinetics ,Cysteine desulfurase activity ,Genes, Bacterial ,biology.protein ,bacteria ,Nif regulon ,ISCU ,Carrier Proteins - Abstract
An IscA homologue within the nif regulon of Azotobacter vinelandii, designated (Nif)IscA, was expressed in Escherichia coli and purified to homogeneity. Purified (Nif)IscA was found to be a homodimer of 11-kDa subunits that contained no metal centers or other prosthetic groups in its as-isolated form. Possible roles for (Nif)IscA in Fe-S cluster biosynthesis were assessed by investigating the ability to bind iron and to assemble Fe-S clusters in a NifS-directed process, as monitored by the combination of UV-vis absorption, Mössbauer, resonance Raman, variable-temperature magnetic circular dichroism, and EPR spectroscopies. Although (Nif)IscA was found to bind ferrous ion in a tetrahedral, predominantly cysteinyl-ligated coordination environment, the low-binding affinity argues against a specific role as a metallochaperone for the delivery of ferrous ion to other Fe-S cluster assembly proteins. Rather, a role for (Nif)IscA as an alternate scaffold protein for Fe-S cluster biosynthesis is proposed, based on the NifS-directed assembly of approximately one labile [4Fe-4S](2+) cluster per (Nif)IscA homodimer, via a transient [2Fe-2S](2+) cluster intermediate. The cluster assembly process was monitored temporally using UV-vis absorption and Mössbauer spectroscopy, and the intermediate [2Fe-2S](2+)-containing species was additionally characterized by resonance Raman spectroscopy. The Mössbauer and resonance Raman properties of the [2Fe-2S](2+) center are consistent with complete cysteinyl ligation. The presence of three conserved cysteine residues in all IscA proteins and the observed cluster stoichiometry of approximately one [2Fe-2S](2+) or one [4Fe-4S](2+) per homodimer suggest that both cluster types are subunit bridging. In addition, (Nif)IscA was shown to couple delivery of iron and sulfur by using ferrous ion to reduce sulfane sulfur. The ability of Fe-S scaffold proteins to couple the delivery of these two toxic and reactive Fe-S cluster precursors is likely to be important for minimizing the cellular concentrations of free ferrous and sulfide ions. On the basis of the spectroscopic and analytical results, mechanistic schemes for NifS-directed cluster assembly on (Nif)IscA are proposed. It is proposed that the IscA family of proteins provide alternative scaffolds to the NifU and IscU proteins for mediating nif-specific and general Fe-S cluster assembly.
- Published
- 2001
8. NifS-Mediated Assembly of [4Fe−4S] Clusters in the N- and C-Terminal Domains of the NifU Scaffold Protein
- Author
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Smith, Archer D., primary, Jameson, Guy N. L., additional, Dos Santos, Patricia C., additional, Agar, Jeffrey N., additional, Naik, Sunil, additional, Krebs, Carsten, additional, Frazzon, Jeverson, additional, Dean, Dennis R., additional, Huynh, Boi Hanh, additional, and Johnson, Michael K., additional
- Published
- 2005
- Full Text
- View/download PDF
9. IscA, an Alternate Scaffold for Fe−S Cluster Biosynthesis
- Author
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Krebs, Carsten, primary, Agar, Jeffrey N., additional, Smith, Archer D., additional, Frazzon, Jeverson, additional, Dean, Dennis R., additional, Huynh, Boi Hanh, additional, and Johnson, Michael K., additional
- Published
- 2001
- Full Text
- View/download PDF
10. Function of conserved tryptophans in the Aspergillus niger glucoamylase 1 starch binding domain.
- Author
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Williamson MP, Le Gal-Coëffet MF, Sorimachi K, Furniss CS, Archer DB, and Williamson G
- Subjects
- Binding Sites, Cyclodextrins metabolism, Glucan 1,4-alpha-Glucosidase genetics, Glucan 1,4-alpha-Glucosidase metabolism, Magnetic Resonance Spectroscopy, Mathematics, Mutagenesis, Site-Directed, Polymerase Chain Reaction, Spectrophotometry, Ultraviolet, Structure-Activity Relationship, Thermodynamics, Aspergillus niger enzymology, Glucan 1,4-alpha-Glucosidase chemistry, Starch metabolism, Tryptophan, beta-Cyclodextrins
- Abstract
Nuclear magnetic resonance (NMR) and ultraviolet (UV) difference spectroscopy were used to assess the role of a number of tryptophan residues in the granular starch binding domain (SBD) of glucoamylase 1 from Aspergillus niger. Wild-type SBD and three variant (W563K, W590K, and W615K) proteins were produced using an A. niger expression system. Titration studies were conducted with beta-cyclodextrin (betaCD), a cyclic analogue of starch, as the ligand. The NMR studies show that the W563K and W590K variants only bind 1 equiv while the wild-type protein forms a 2:1 (ligand:protein) complex. It also clearly demonstrates the abolition of binding at site 1 and site 2 in W590K and W563K, respectively. UV difference spectroscopy was used to calculate dissociation constants with addition of betaCD: 14.4 microM (apparent) for the wild type, 28.0 microM for W563K, and 6.4 microM for W590K. The implication of this is that the two binding sites have unequal contributions to the overall binding of the SBD which may be related to functional differences between the two binding sites. The low stability of the third variant, W615K, suggests that this tryptophan is not involved in binding but has an essential structural role.
- Published
- 1997
- Full Text
- View/download PDF
11. Structural determinants of protein dynamics: analysis of 15N NMR relaxation measurements for main-chain and side-chain nuclei of hen egg white lysozyme.
- Author
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Buck M, Boyd J, Redfield C, MacKenzie DA, Jeenes DJ, Archer DB, and Dobson CM
- Subjects
- Amino Acid Sequence, Animals, Asparagine, Chickens, Crystallography, X-Ray, Female, Glutamine, Magnetic Resonance Spectroscopy methods, Models, Molecular, Muramidase biosynthesis, Nitrogen Isotopes, Recombinant Fusion Proteins biosynthesis, Recombinant Proteins chemistry, Thermodynamics, Muramidase chemistry, Protein Structure, Secondary
- Abstract
15N-labeled hen lysozyme has been studied by 2D and 3D NMR in order to characterize its dynamic behavior. The resonances of all main-chain amide nitrogen atoms were assigned, as were resonances of nitrogen atoms in 28 side chains. Relaxation measurements for the main-chain and arginine and tryptophan side-chain 15N nuclei used standard methods, and those for the 15N nuclei of asparagine and glutamine side chains used pulse sequences designed to remove unwanted relaxation pathways in the NH2 groups. The calculated order parameters (S2) show that the majority of main-chain amides undergo only small amplitude librational motions on a fast time scale (S2 > or = 0.8). Increased main-chain motion (0.5 < S2 < 0.8) is observed for a total of 19 residues located at the C-terminus, in loop and turn regions, and in the first strand of the main beta-sheet. Order parameters derived for the side chains range from 0.05 to 0.9; five of the six tryptophan residues have high order parameters (S2 > or = 0.8), consistent with their location in the closely packed core of the protein, whereas the order parameters between 0.05 and 0.3 for arginine residues confirm increased side-chain mobility at the protein surface. Order parameters for the side chains of asparagine and glutamine residues range from 0.2 to 0.8; high values are found for side chains that have low solvent accessible surfaces and well-defined chi 1 values, as measured by 3J alpha beta coupling constants. Many of the main-chain and side-chain groups with low order parameters have higher than average temperature factors in X-ray crystal structures and increased positional uncertainty in NMR solution structures. They also tend to lack persistent hydrogen bond interactions and protection against amide hydrogen exchange. The most significant correlations are found between residues with low order parameters and high surface accessibility in both crystal and solution structures. The results suggest that a lack of van der Waals contacts is a major determinant of side-chain and main-chain mobility in proteins.
- Published
- 1995
- Full Text
- View/download PDF
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