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PubMed1989

Protein structure alignment.

A new method of comparing protein structures is described, based on distance plot analysis. It is relatively insensitive to insertions and deletions in sequence and is tolerant of the displacement of equivalent substructures between the two molecules being compared. When presented with the co-ordinate sets of two structures, the method will produce automatically an alignment of their sequences based on structural criteria. The method uses the dynamic programming optimization technique, which is widely used in the comparison of protein sequences and thus unifies the techniques of protein structure and sequence comparison. Typical structure comparison problems were examined and the results of the new method compared to the published results obtained using conventional methods. In most examples, the new method produced a result that was equivalent, and in some cases superior, to those reported in the literature.

Algorithms↗
PubMed1991

Preliminary crystallographic study of a pseudoazurin from methylotrophic bacterium, Methylobacterium extorquens AM1.

Single crystals of pseudoazurin, one of the blue copper proteins produced by methylotrophic bacterium Methylobacterium extorquens AM1, have been obtained by the method of vapor diffusion with ammonium sulfate as a precipitant at pH 8.0. Crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit cell dimensions of a = 52.619(4) A, b = 63.280(6) A, c = 35.133(4) A. The asymmetric unit includes one molecule of pseudoazurin (Vm = 2.18 A3/dalton). The crystals are so stable against X-ray irradiation that diffraction intensities of the native crystal up to 1.68 A resolution could be collected from only one crystal. Among the many heavy-metal reagents examined, uranyl acetate gave an effective isomorphous derivative.

Azurin↗
PubMed1992

Crystallization and preliminary X-ray crystallographic studies of rusticyanin from Thiobacillus ferrooxidans.

Rusticyanin is a 16.5 kDa type I blue copper protein isolated from Thiobacillus ferrooxidans. This organism can grow on Fe2+ as its sole energy source. Rusticyanin is thought to be a principal component in the iron respiratory electron transport chain of T. ferrooxidans. As a component of the periplasmic space of an acidophilic bacterium, rusticyanin is remarkably stable at acidic pH. It is redox-active down to pH 0.2. Crystals of rusticyanin have been grown from solutions of PEG 8000 by the hanging-drop vapor diffusion method. The crystals are orthorhombic, space group P2(1)2(1)2(1), with unit cell dimensions a = 32.36 A, b = 60.37 A, c = 74.60 A. The crystals diffract to 2.0 A resolution and they are stable in the X-ray beam for at least two days.

Azurin↗
PubMed1994

Volume changes in protein evolution.

We have determined the variations in volume that occur during evolution in the buried core of three different families of proteins. The variation of the whole core is very small (approximately 2.5%) compared to the variation at individual sites (approximately 13%). However, by comparing our results to those expected from random sequences with no correlations between sites, we show that the small variation observed may simply be a manifestation of the statistical "law of large numbers" and not reflect any compensating changes in, or global constraints upon, protein sequences. We have also analysed in detail the volume variations at individual sites, both in the core and on the surface, and compared these variations with those expected from random sequences. Individual sites on the surface have nearly the same variation as random sequences (24% versus 28% variation). However, individual sites in the core have about half the variation of random sequences (13% versus 30%). Roughly, half of these core sites strongly conserve their volume (0 to 10% variation); one quarter have moderate variation (10 to 20%); and the remaining quarter vary randomly (20 to 40%). Our results have clear implications for the relationship between protein sequence and structure. For our analysis, we have developed a new and simple method for weighting protein sequences to correct for unequal representation, which we describe in an Appendix.

Algorithms↗
PubMed1986

Preferences of the side chains in proteins for helix, beta strand, turn, and other conformations. Secondary structures of copper proteins.

Statistical preferences of the side chains in proteins for the helical conformation, the beta strand conformation, and the turn conformation were derived from the computer-assigned secondary structures of 55 protein chains reported by Levitt and Greer in 1977 after averaging redundant structures and weighting structures that showed greater than 25% homology by w = square root N/N. Separate preferences are reported for free cysteine side chains and for cystine. These preferences are used to predict the secondary structures of cucumber plastocyanin and cupredoxin, whose crystal structures will be reported soon. The loops in the C-terminal copper-binding regions are discussed.

Amino Acid Sequence↗
PubMed1993

Amino-acid sequence of rusticyanin from Thiobacillus ferrooxidans and its comparison with other blue copper proteins.

Rusticyanin, a copper protein characterized by a high redox potential (+680 mV) and a high stability at acidic pH, is involved in iron oxidation in Thiobacillus ferrooxidans. It has been characterized from a new strain and its amino-acid sequence has been determined and compared to two other rusticyanin sequences isolated from different strains. It comprises 155 amino acids and the alignment of the three rusticyanins shows a high degree of homology. Comparing the rusticyanins with six blue copper proteins which have a copper-I site in common, a consensus sequence containing Cys, His and Met in the C-terminal part of the protein and His-85 is proposed to be involved in the copper coordination. Secondary structure predictions are compared to three structures of copper proteins obtained by X-ray crystallography.

Amino Acid Sequence↗
PubMed1996

Mavicyanin, a stellacyanin-like protein from zucchini peelings: primary structure and comparison with other cupredoxins.

The complete amino-acid sequence of mavicyanin, a small blue copper-containing glycoprotein isolated from zucchini peelings, is presented. The sequence of this cupredoxin was deduced from analysis of peptides obtained after cleavage of the protein with trypsin or Asp-N endoproteinase. Mavicyanin consists of a single polypeptide chain of 108 amino-acid residues. Accurate molecular weight determination by electrospray mass spectrometry (12 752 Da) indicates a mass difference of approx. 1005 Da with respect to the mass of the protein, as determined on the basis of the amino-acid sequence (11747 Da). This difference was tentatively assigned to the carbohydrate moiety, not yet characterized, attached to the protein via an N-linkage to Asn-58 and O-linkages to unidentified Ser/Thr residues. The comparison of the primary structure of mavicyanin with those of other cupredoxins shows that three copper ligands (His-44, Cys-57 and His-90) are conserved, while a glutamine residue (Gln-95), as in stellacyanin, is possibly the fourth ligand. An amino-acid sequence alignment of mavicyanin with copper proteins currently identified as phytocyanins is also proposed, showing same invariant residues in key positions related to the maintenance of the beta-barrel fold and to the active site.

Amino Acid Sequence↗
PubMed1984

Denitrification and nitrite reduction: Pseudomonas aeruginosa nitrite-reductase.

Present knowledge of the different enzymatic steps of the denitrification chains in various bacteria, particularly Paracoccus denitrificans and Pseudomonas aeruginosa has been briefly reviewed. The question whether nitric oxide (NO), nitrous oxide (N2O) and other nitrogen derivatives are obligatory intermediates has been discussed. The second part is an extensive review of the structure and the function of a key enzyme in denitrification, cytochrome c551-nitrite-oxidoreductase from P. aeruginosa. Recent results on the stoichiometry of nitrite reduction have been discussed.

Amino Acids↗
PubMed1993

A negatively light-regulated gene from Arabidopsis thaliana encodes a protein showing high similarity to blue copper-binding proteins.

A negatively photo-regulated gene (bcb) has been isolated by means of differential hybridization of a genomic library of Arabidopsis thaliana. In mature plants, a 20-fold increase in the amount of steady-state bcb mRNA can be detected upon 48 h of dark adaption. The expression level of the gene is also dependent upon the developmental stage of the plant. The 21.5-kDa gene product (BCB) shows extensive similarity with blue Cu(2+)-binding proteins such as plastocyanin and stellacyanin. The protein abundance increases only twofold upon dark adaption, which implies the presence of post-transcriptional control. The isolation of a novel negatively photoregulated gene allows us to investigate the complex expression profile of genes responding to the absence of light.

Amino Acid Sequence↗
PubMed1995

Depletion and replacement of protein metal ligands.

Recently, site-directed mutagenesis has been applied to protein-derived metal ligands in a way that permits the replacement in trans of protein ligands. The chemical diversity of ligands available using this method far exceeds that attainable using standard mutagenesis. Non-conservative ligand replacement can yield novel metalloproteins with altered ligand-binding, enzymatic activities, and spectroscopic properties. Conservative ligand substitution, or 'ligand detachment', allows the structural and functional effects of the covalent linkage between the ligand and the protein to be evaluated; this linkage is often proposed to play a critical role in modulating the structure and reactivity of the metal center. Furthermore, this method can be exploited to study the details of molecular recognition at the structural, thermodynamic, and dynamic levels.

Azurin↗
PubMed2005

Rapid binding of copper(I) to folded aporusticyanin.

Kinetics of copper uptake in both oxidation states by the folded and unfolded forms of the type 1 copper protein rusticyanin have been studied. The speed of the binding of copper(I) to the folded rusticyanin is fast, and of the same order of magnitude as copper(I) uptake by the unfolded form. Thus, the binding of copper can be subsequent to the protein folding, contrary to previous proposals. Implications for the mechanism of the formation of the active holoprotein in vivo are discussed.

Azurin↗
PubMed2006

Using metal-catalyzed oxidation reactions and mass spectrometry to identify amino acid residues within 10 A of the metal in Cu-binding proteins.

Metal-catalyzed oxidation (MCO) reactions and mass spectrometry (MS) can be used together to determine the amino acids bound to Cu in a metalloprotein. Selective oxidation of only amino acids bound to Cu during the MCO/MS approach relies on proper choice of the types and concentrations of the reducing and oxidizing agents. We show here that if these MCO reagent concentrations are "detuned" or varied slightly from optimal conditions, nonmetal-bound amino acids close to Cu can also be oxidized in a controlled manner. Using Cu/Zn superoxide dismutase as a model system, we demonstrate that amino acids up to 10 A from Cu can be modified as long as they are readily accessible to diffusing reactive oxygen species. UV/VIS spectroscopy and measurements of oxidation kinetics provide evidence that the protein's structural integrity around Cu is maintained during the detuned MCO reactions. Because this method can identify amino acids around Cu that have low solvent accessibility, it should complement other radical-based protein surface-mapping techniques.

Amino Acids↗
PubMed2004

Interpretation of the temperature-dependent color of blue copper protein mutants.

The electronic absorption spectrum of the mutant of the blue copper protein amicyanin with a pseudoazurin loop (AmiPse) shows a remarkable temperature dependence. The absorption band at approximately 460 nm increases at low temperature while the transition at approximately 600 nm is not much affected by a variation of the temperature. An approximate density functional theory (DFT) study of the active site model [Cu(II)(imidazole)(2)(SCH(3))(S(CH(3))(2))](+) (protein backbone and solvation neglected) leads to two local minimum structures (axial and rhomb) which both have a geometry close to that typical for blue copper proteins. One (rhomb) has two structurally different histidine donors, and this geometry is also found in most experimental type 1 structures. The two forms axial and rhomb are distortional isomers and are energetically almost degenerate. The temperature dependence of the spectrum of AmiPse is interpreted with a temperature-dependent change of the relative population of the two local minimum structures with slightly different energy. The 460 nm transition is believed to be due to preferential population of the structure rhomb; this is in agreement with the published assignment of the high energy transition, based on thorough spectroscopic and computational studies. Consequences of a perturbation of the "gas phase" structures axial and rhomb by the protein and solvation are also discussed on the basis of published, experimentally observed structures and spectroscopic data.

Azurin↗
PubMed2006

Modelling the impact of geometric parameters on the redox potential of blue copper proteins.

The synthesis and structure of a homologous series of cationic N(2)S(2) copper(I) Schiff base complexes constructed using o-tert-butylthiobenzaldehyde and a series of terminal diamines (ethane, propane, butane) are reported. The complexes differ only in the length of the methylene chain between the imine groups. This simple modification forces the copper centre to shift geometry from a planar (1,2-diaminoethane) to a more distorted tetrahedral motif (1,4-diaminobutane). The redox potentials of the three cations were measured using cyclic voltammetry in donor (acetonitrile) and non-donor solvents (dichloromethane). The S-Cu-N angles for each complex are correlated against the respective redox potential allowing an analysis of the geometric impact on the redox potential in soft copper centres. The redox potential is observed to increase as the metal centre moves from a planar towards a tetrahedral motif. Comparing this data with the reported structures of the blue copper proteins (rusticyanin and plastocyanin) allows an assessment of the contribution of the geometry of the metal binding site to the operating potential of these proteins to be made.

Azurin↗
PubMed2006

DNA cleavage mediated by copper superoxide dismutase via two pathways.

The known action of Cu, Zn superoxide dismutase (Cu(2)Zn(2)SOD) that converts O(2)(-) to O(2) and H(2)O(2) plays a crucial role in protecting cells from toxicity of oxidative stress. However, the overproduction of Cu(2)Zn(2)SOD does not result in increased protection but rather creates a variety of unfavorable effects, suggesting that too much Cu(2)Zn(2)SOD may be injurious to the cells. The present study examined the DNA cleavage activity mediated by a Cu(n)SOD that contains 1-4 copper ions, in order to obtain an insight into the aberrant copper-mediated oxidative chemistry in the enzyme. A high SOD activity was observed upon metallation of the apo-form of Cu(2)Zn(2)SOD with Cu(II), indicating that nearly all of the Cu(II) in the Cu(n)SOD is as active as the Cu(II) in the copper site of fully active Cu(2)Zn(2)SOD. Using a supercoiled DNA as substrate, significant DNA cleavage was observed with the Cu(n)SOD in the presence of hydrogen peroxide or mercaptoethanol, whereas DNA cleavage with free Cu(II) ions can occur only <5% under the same conditions. Comparison with other proteins shows that the DNA cleavage activity is specific to some proteins including the Cu(n)SOD. The steady state study suggests that a cooperative action between the SOD protein and the Cu(II)may appear in the DNA cleavage activity, which is independent of the number of Cu(II) in the Cu(n)SOD. The kinetic study shows that a two-stage reaction was involved in DNA cleavage. The effects of various factors including EDTA, radical scavengers, bicarbonate anion, and carbon dioxide gas molecules on the Cu(n)SOD-mediated DNA cleavage activity were also investigated. It is proposed that DNA cleavage occurs via both hydroxyl radical oxidation and hydroxide ion hydrolysis pathways. This work implies that any form of the copper-containing SOD enzymes (including Cu(2)Zn(2)SOD and its mutants) might have the DNA cleavage activity.

Animals↗
PubMed2006

Structural diversity of domain superfamilies in the CATH database.

The CATH database of domain structures has been used to explore the structural variation of homologous domains in 294 well populated domain structure superfamilies, each containing at least three sequence diverse relatives. Our analyses confirm some previously detected trends relating sequence divergence to structural variation but for a much larger dataset and in some superfamilies the new data reveal exceptional structural variation. Use of a new algorithm (2DSEC) to analyse variability in secondary structure compositions across a superfamily sheds new light on how structures evolve. 2DSEC detects inserted secondary structures that embellish the core of conserved secondary structures found throughout the superfamily. Analysis showed that for 56% of highly populated superfamilies (>9 sequence diverse relatives), there are twofold or more increases in the numbers of secondary structures in some relatives. In some families fivefold increases occur, sometimes modifying the fold of the domain. Manual inspection of secondary structure insertions or embellishments in 48 particularly variable superfamilies revealed that although these insertions were usually discontiguous in the sequence they were often co-located in 3D resulting in a larger structural motif that often modified the geometry of the active site or the surface conformation promoting diverse domain partnerships and protein interactions. These observations, supported by automatic analysis of all well populated CATH families, suggest that accretion of small secondary structure insertions may provide a simple mechanism for evolving new functions in diverse relatives. Some layered domain architectures (e.g. mainly-beta and alpha-beta sandwiches) that recur highly in the genomes more frequently exploit these types of embellishments to modify function. In these architectures, aggregation occurs most often at the edges, top or bottom of the beta-sheets. Information on structural variability across domain superfamilies has been made available through the CATH Dictionary of Homologous Structures (DHS).

Amino Acid Sequence↗