Small Copper Protein Research Articles

Modules Identification in Protein Structures: The Topological and Geometrical Solutions

The identification of modules in protein structures has major relevance in structural biology, with consequences in protein stability and functional classification, adding new perspectives in drug design. In this work, we present the comparison between a topological (spectral clustering) and a geometrical (k-means) approach to module identification, in the frame of a multiscale analysis of the protein architecture principles. The global consistency of an adjacency matrix based technique (spectral clustering) and a method based on full rank geometrical information (k-means) give a proof-of-concept of the relevance of protein contact networks in structure determination. The peculiar "small-world" character of protein contact graphs is established as well, pointing to average shortest path as a mesoscopic crucial variable to maximize the efficiency of within-molecule signal transmission. The specific nature of protein architecture indicates topological approach as the most proper one to highlight protein functional domains, and two new representations linking sequence and topological role of aminoacids are demonstrated to be of use for protein structural analysis. Here we present a case study regarding azurin, a small copper protein implied in the Pseudomonas aeruginosa respiratory chain. Its pocket molecular shape and its electron transfer function have challenged the method, highlighting its potentiality to catch jointly the structure and function features of protein structures through their decomposition into modules.

Mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by P. pantotrophus pseudoazurin: kinetics of intermolecular electron transfer

This work reports the direct electrochemistry of Paracoccus pantotrophus pseudoazurin and the mediated catalysis of cytochrome c peroxidase from the same organism. The voltammetric behaviour was examined at a gold membrane electrode, and the studies were performed in the presence of calcium to enable the peroxidase activation. A formal reduction potential, E (0)', of 230 +/- 5 mV was determined for pseudoazurin at pH 7.0. Its voltammetric signal presented a pH dependence, defined by pK values of 6.5 and 10.5 in the oxidised state and 7.2 in the reduced state, and was constant up to 1 M NaCl. This small copper protein was shown to be competent as an electron donor to cytochrome c peroxidase and the kinetics of intermolecular electron transfer was analysed. A second-order rate constant of 1.4 +/- 0.2 x 10(5) M(-1) s(-1) was determined at 0 M NaCl. This parameter has a maximum at 0.3 M NaCl and is pH-independent between pH 5 and 9.

Conformational Changes in Azurin from Pseudomona aeruginosa Induced through Chemical and Physical Protocols

Azurin from Pseudomona aeruginosa is a small copper protein with a single tryptophan (Trp) buried in the structure. The Gibbs free energies associated with the folding of holo azurin, calculated monitoring Trp fluorescence and changes in absorbance on the ligand-to-metal band, are different because these techniques probe their local environments, thereby being able to probe different conformational changes. The presence of an intermediate state was observed during the chemical denaturation of the protein. Upon denaturation, a 30-fold increase is observed in the magnitude of the quenching constant of the tryptophan fluorescence by acrylamide, because this residue becomes more accessible to the quencher. Entrapping the protein in sol-gel materials lowers its stability possibly because the solvation properties of the macromolecule are changed. The thermal denaturation of azurin immobilized in a sol-gel monolith is irreversible, which tends to rule out an aggregation mechanism to account for the irreversibility of the denaturation of the protein free in solution. Unlike the Cu(II) ion, the Gd(III) ion accommodates in site B of azurin with high affinity and the folding free energy of Gd-azurin is larger than that of apo azurin.

Auracyanin A from the thermophilic green gliding photosynthetic bacterium Chloroflexus aurantiacus represents an unusual class of small blue copper proteins.

The amino acid sequence of the small copper protein auracyanin A isolated from the thermophilic photosynthetic green bacterium Chloroflexus aurantiacus has been determined to be a polypeptide of 139 residues. His58, Cys123, His128, and Met132 are spaced in a way to be expected if they are the evolutionary conserved metal ligands as in the known small copper proteins plastocyanin and azurin. Secondary structure prediction also indicates that auracyanin has a general beta-barrel structure similar to that of azurin from Pseudomonas aeruginosa and plastocyanin from poplar leaves. However, auracyanin appears to have sequence characteristics of both small copper protein sequence classes. The overall similarity with a consensus sequence of azurin is roughly the same as that with a consensus sequence of plastocyanin, namely 30.5%. We suggest that auracyanin A, together with the B forms, is the first example of a new class of small copper proteins that may be descendants of an ancestral sequence to both the azurin proteins occurring in prokaryotic nonphotosynthetic bacteria and the plastocyanin proteins occurring in both prokaryotic cyanobacteria and eukaryotic algae and plants. The N-terminal sequence region 1-18 of auracyanin is remarkably rich in glycine and hydroxy amino acids, and required mass spectrometric analysis to be determined. The nature of the blocking group X is not yet known, although its mass has been determined to be 220 Da. The auracyanins are the first small blue copper proteins found and studied in anoxygenic photosynthetic bacteria and are likely to mediate electron transfer between the cytochrome bc1 complex and the photosynthetic reaction center.

The amino acid sequence of a type I copper protein with an unusual serine- and hydroxyproline-rich C-terminal domain isolated from cucumber peelings

We have determined the amino acid sequence of a small copper protein isolated from cucumber peelings. This cupredoxin contains 137 amino acids including a pyroglutamate as the first residue. The N-terminal 110 amino acid-long domain shows 30–37% identity to 2 other cupredoxins, stellacyanin and cucumber basic blue protein. A unique feature of this protein is a 27 amino acid-long, C-terminal domain rich in 4-hydroxyproline and serine and resembling certain plant cell wall proteins. The prolines in this domain are hydroxylated to a different extent depending on the surrounding sequence.

A two-dimensional NMR method for assignment of imidazole ring proton resonances of histidine residues in proteins

The use of two-dimensional scalar correlated NMR spectroscopy for the assignment of histidine imidazole ring proton resonances is described. The method allows unequivocal assignment of both the C-2 and C-4 proton resonances belonging to individual histidine residues. Applications to the small copper protein, plastocyanin, and to myoglobin, a protein containing many histidines, are described. This method should be generally applicable to histidine-containing proteins of moderate size.

Structural and Molecular Weight Studies on the Small Copper Protein, Plastocyanin

Abstract The plastocyanin from Phaseolus vulgaris has been shown to consist of a single polypeptide chain of 100 residues to which 1 atom of copper is bound. This plastocyanin is the smallest copper-protein yet reported; its molecular weight is 10,690. The molar extinction coefficient (e597) of the copper chromophore was shown to be 4.5 x 103. The copper content and molecular weight contrast with that of plastocyanin preparations from spinach (mol. wt., 21,000) and Chenopodium album (mol. wt., 11,500), which both contain 2 copper atoms (2, 13). Amino acid analysis indicated that the minimum molecular weight of the polypeptide chain is 10,626. The dansyl-Edman procedure showed that plastocyanin has only the one NH2-terminal sequence, Leu-Glx-Val-Leu, and reduction and carboxymethylation of the apoprotein gave a homogeneous product, as judged by acrylamide gel electrophoresis and Sephadex gel chromatography. The molecular weight of the carboxymethyl protein was similar to the minimum molecular weight value of 10,626. There are 2 residues of methionine per minimum molecular weight, and cyanogen bromide cleavage of the carboxymethyl protein gave 3 fragments, containing 58, 35, and 7 residues. The characterization of these peptides confirmed that the protein contains only one type of polypeptide chain, with a molecular weight of 10,626. Molecular weight values for the native protein of about 11,000 were consistently obtained by Sephadex gel chromatography in sodium acetate buffers at pH 6.0 and of varying ionic strengths. The approach to equilibrium technique yielded a value of 10,790 in sodium acetate buffer, pH 6.0. Variable and high molecular weight values for plastocyanin (up to 16,000) were obtained by Sephadex gel chromatography in sodium phosphate buffers. The reason for these anomalous values is not clear. Frontal analysis studies with Sephadex did not indicate dimerization of the molecule in the phosphate buffer. There was no significant difference in the molecular weight or sedimentation coefficient of plastocyanin in acetate and phosphate buffers as judged by ultracentrifugation.