A partially folded intermediate species of the beta-sheet protein apo-pseudoazurin is trapped during proline-limited folding. (25/282)

The folding of apo-pseudoazurin, a 123-residue, predominantly beta-sheet protein with a complex Greek key topology, has been investigated using several biophysical techniques. Kinetic analysis of refolding using far- and near-ultraviolet circular dichroism (UV CD) shows that the protein folds slowly to the native state with rate constants of 0.04 and 0.03 min(-1), respectively, at pH 7.0 and at 15 degrees C. This process has an activation enthalpy of approximately 90 kJ/mole and is catalyzed by cyclophilin A, indicating that folding is limited by trans-cis proline isomerization, presumably around the Xaa-Pro 20 bond that is in the cis isomer in the native state. Before proline isomerization, an intermediate accumulates during folding. This species has a substantial signal in the far-UV CD, a nonnative signal in the near-UV CD, exposed hydrophobic surfaces (judged by 1-anilino naphthalenesulphonate binding), a noncooperative denaturation transition, and a dynamic structure (revealed by line broadening on the nuclear magnetic resonance time scale). We compare the properties of this intermediate with partially folded states of other proteins and discuss its role in folding of this complex Greek key protein.  (+info)

A novel C1-using denitrifier alcaligenes sp. STC1 and its genes for copper-containing nitrite reductase and azurin. (26/282)

A novel denitrifier Alcaligenes sp. STC1 was identified. The strain efficiently denitrifies under an atmosphere of 10% oxygen (O2) where Paracoccus denitrificans, one of the most studied aerobic denitrifiers, had less denitrifying activity, indicating that the strain has an O2-torelant denitrifying system. It denitrified by using C1-carbon sources such as formate and methanol as well as glucose, glycerol, and succinate. The genes for the copper-containing nitrite reductase and azurin of this C1-using denitrifier were cloned. Their predicted products of them were similar to those of their counterparts and the maximum similarities were 90% and 92%, respectively.  (+info)

Azurin involved in alcohol oxidation system in Pseudomonas putida HK5: expression analysis and gene cloning. (27/282)

Expression of azurin in Pseudomonas putida HK5 was examined by immunoblot analysis. Similar amounts of azurin were found in the cells grown into the stationary phase on any carbon sources, including LB medium without alcohol, where no quinoprotein alcohol dehydrogenases appeared. In the early exponential phase, the highest amount of azurin was found in the cells grown on 1-butanol, but here was none in the case of LB medium, suggesting that expression of azurin is cooperative with that of the alcohol oxidase system, especially the system including quinohemoprotein alcohol dehydrogenase IIB. The azurin gene (azu) was cloned and sequenced. azu is monocistronic, and in its promoter region, FNR-binding consensus sequence was found. However, its relative position suggests different transcriptional regulation from that in azu of P. aeruginosa. The molecular weight of the mature protein without copper ion calculated from the amino acid sequence was consistent with the value of the purified azurin measured by mass spectrometry.  (+info)

Characterization of nirV and a gene encoding a novel pseudoazurin in Rhodobacter sphaeroides 2.4.3. (28/282)

Sequencing of the region flanking nirK, the gene encoding the copper-containing nitrite reductase in Rhodobacter sphaeroides 2.4.3, has identified two genes whose products could potentially be involved in nitrite reductase expression and activity. One of the genes has been designated nirV. Putative nirV orthologues are found in other denitrifiers, where they are also located downstream of the structural gene for nitrite reductase. The nirV in 2.4.3 is apparently cotranscribed with nirK. Inactivation of nirV had no effect on cell growth, or on nitrite reductase expression or activity. Downstream of nirV and divergently transcribed is a gene, designated ppaZ, encoding a protein with significant similarity to pseudoazurins from other denitrifiers. However, three of the four residues required for binding of the type I copper centre are not conserved in the deduced sequence of the protein in 2.4.3. ppaZ is expressed only when oxygen becomes limiting. ppaZ expression is dependent on both FnrL and NnrR, and a putative binding site for these proteins has been identified. Expression of ppaZ is also dependent on the two-component PrrB/PrrA system. Inactivation of ppaZ had no significant effect on cell growth or on nitrite reductase expression or activity. Expression of a maltose-binding protein-PpaZ fusion indicated that the protein could not bind copper. Examination of the genome of the related bacterium R. sphaeroides 2.4.1 revealed that it encodes ppaZ but not nirV and evidence is presented suggesting that a common ancestor of 2.4.3 and 2.4.1 had both nitrite and nitric oxide reductase activity but as the strains diverged 2.4.1 lost nirK and nirV, making it incapable of nitrite reduction.  (+info)

Studies of Pseudomonas aeruginosa azurin mutants: cavities in beta-barrel do not affect refolding speed. (29/282)

Pseudomonas aeruginosa azurin is a blue-copper protein with a Greek-key fold. Removal of copper produces an apoprotein with the same structure as holoazurin. To address the effects on thermodynamic stability and folding dynamics caused by small cavities in a beta-barrel, we have studied the behavior of the apo-forms of wild-type and two mutant (His-46-Gly and His-117-Gly) azurins. The equilibrium- and kinetic-folding and unfolding reactions appear as two-state processes for all three proteins. The thermodynamic stability of the two mutants is significantly decreased as compared with the stability of wild-type azurin, in accord with cavities in or near the hydrophobic interior having an overall destabilizing effect. Large differences are also found in the unfolding rates: the mutants unfold much faster than wild-type azurin. In contrast, the folding-rate constants are almost identical for the three proteins and closely match the rate-constant predicted from the native-state topology of azurin. We conclude that the topology is more important than equilibrium stability in determining the folding speed of azurin.  (+info)

Role of the coordinating histidine in altering the mixed valency of Cu(A): an electron nuclear double resonance-electron paramagnetic resonance investigation. (30/282)

The binuclear Cu(A) site engineered into Pseudomonas aeruginosa azurin has provided a Cu(A)-azurin with a well-defined crystal structure and a CuSSCu core having two equatorial histidine ligands, His120 and His46. The mutations His120Asn and His120Gly were made at the equatorial His120 ligand to understand the histidine-related modulation to Cu(A), notably to the valence delocalization over the CuSSCu core. For these His120 mutants Q-band electron nuclear double resonance (ENDOR) and multifrequency electron paramagnetic resonance (EPR) (X, C, and S-band), all carried out under comparable cryogenic conditions, have provided markedly different electronic measures of the mutation-induced change. Q-band ENDOR of cysteine C(beta) protons, of weakly dipolar-coupled protons, and of the remaining His46 nitrogen ligand provided hyperfine couplings that were like those of other binuclear mixed-valence Cu(A) systems and were essentially unperturbed by the mutation at His120. The ENDOR findings imply that the Cu(A) core electronic structure remains unchanged by the His120 mutation. On the other hand, multifrequency EPR indicated that the H120N and H120G mutations had changed the EPR hyperfine signature from a 7-line to a 4-line pattern, consistent with trapped-valence, Type 1 mononuclear copper. The multifrequency EPR data imply that the electron spin had become localized on one copper by the His120 mutation. To reconcile the EPR and ENDOR findings for the His120 mutants requires that either: if valence localization to one copper has occurred, the spin density on the cysteine sulfurs and the remaining histidine (His46) must remain as it was for a delocalized binuclear Cu(A) center, or if valence delocalization persists, the hyperfine coupling for one copper must markedly diminish while the overall spin distribution on the CuSSCu core is preserved.  (+info)

Crystal structure of the double azurin mutant Cys3Ser/Ser100Pro from Pseudomonas aeruginosa at 1.8 A resolution: its folding-unfolding energy and unfolding kinetics. (31/282)

Azurin is a cupredoxin, which functions as an electron carrier. Its fold is dominated by a beta-sheet structure. In the present study, azurin serves as a model system to investigate the importance of a conserved disulphide bond for protein stability and folding/unfolding. For this purpose, we have examined two azurin mutants, the single mutant Cys3Ser, which disrupts azurin's conserved disulphide bond, and the double mutant Cys3Ser/Ser100Pro, which contains an additional mutation at a site distant from the conserved disulphide. The crystal structure of the azurin double mutant has been determined to 1.8 A resolution(2), with a crystallographic R-factor of 17.5% (R(free)=20.8%). A comparison with the wild-type structure reveals that structural differences are limited to the sites of the mutations. Also, the rates of folding and unfolding as determined by CD and fluorescence spectroscopy are almost unchanged. The main difference to wild-type azurin is a destabilisation by approximately 20 kJ x mol(-1), constituting half the total folding energy of the wild-type protein. Thus, the disulphide bond constitutes a vital component in giving azurin its stable fold.  (+info)

Structure at 1.9 A resolution of a quinohemoprotein alcohol dehydrogenase from Pseudomonas putida HK5. (32/282)

The type II quinohemoprotein alcohol dehydrogenase of Pseudomonas putida is a periplasmic enzyme that oxidizes substrate alcohols to the aldehyde and transfers electrons first to pyrroloquinoline quinone (PQQ) and then to an internal heme group. The 1.9 A resolution crystal structure reveals that the enzyme contains a large N-terminal eight-stranded beta propeller domain (approximately 60 kDa) similar to methanol dehydrogenase and a small C-terminal c-type cytochrome domain (approximately 10 kDa) similar to the cytochrome subunit of p-cresol methylhydoxylase. The PQQ is bound near the axis of the propeller domain about 14 A from the heme. A molecule of acetone, the product of the oxidation of isopropanol present during crystallization, appears to be bound in the active site cavity.  (+info)