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PubMed1995

Structural and spectroscopic studies of the copper site of stellacyanin.

The structure of the copper site in oxidized and reduced Rhus vernicifera stellacyanin has been studied by X-ray absorption (XAFS) spectroscopy at different pH values. Data for the oxidized protein are consistent with the fourth ligand being an O- or N-donating ligand rather than a cysteine from the disulfide bridge. The fourth ligand is not present in the inner coordination sphere, but makes a more distant interaction 2.7 A from the copper atom. Only minor changes in the details of the Cu(II) coordination occur when the pH is varied. Direct structural information on reduced stellacyanin is provided. Upon reduction, one of the histidine ligands moves away from the copper atom by at least 0.2 A. A low-Z (O or N) scatterer is present approximately 2.4 A from the Cu(I) atom in the protein at low pH, and this ligand is lost at high pH. There is no evidence for an S-donating fourth ligand in the reduced protein. The XAFS results are presented in relation to the spectroscopic and structural information available for some methionine-121 mutants of azurin. The data reveal that there are spectroscopic similarities between stellacyanin and some of the mutant proteins, but distinct structural differences exist that preclude these proteins as suitable models for the copper site of stellacyanin.

Azurin↗
PubMed2001

Electronic characterization of the oxidized state of the blue copper protein rusticyanin by 1H NMR: is the axial methionine the dominant influence for the high redox potential?

The oxidized state of rusticyanin, the blue copper protein with the highest redox potential in its class, has been investigated through (1)H nuclear magnetic resonance applied to its cobalt(II) derivative. The assignment of the protons belonging to the coordinated residues has been performed. Many other amino acids situated in the vicinity of the metal ion, including six hydrophobic residues (isoleucine140 and five phenylalanines) have also been identified. The orientation of the main axes of the magnetic susceptibility tensor for the cobalt(II)-rusticyanin as well as its axial, Deltachi(ax), and rhombic, Deltachi(rh), magnetic susceptibility anisotropy components have been determined. A comparison of the present results with those previously obtained for cobalt(II)azurin [Donaire, A., Salgado, J., Moratal, J. M. (1998) Biochemistry 37, 8659-8673] allows us to provide further insights into the reasons for the high redox potential of this protein. According to our results, the interaction between the metal ion and the thioether Sdelta of the axial methionine is not as influential as the strong destabilizing effect that the hydrophobic residues close to the metal ion undergo in the oxidized state.

Anisotropy↗
PubMed1994

Raman spectroscopic study of the blue copper protein halocyanin from Natronobacterium pharaonis.

Halocyanin, a blue copper protein from Natronobacterium pharaonis was studied by Raman spectroscopic techniques. Near-infrared Fourier transform Raman spectra, which for the first time have been employed to study copper proteins in both oxidation states, display the Raman bands of the protein and, in the oxidized state, also the preresonance-enhanced bands of the copper center. The frequency of the amide I band at 1676 cm-1 indicates a predominant beta-sheet protein structure, which is typical for small blue copper proteins. The Raman spectra provide no evidence for major redox-linked changes of the secondary structure. Resonance Raman spectra of the oxidized protein obtained upon excitation close to the 600-nm absorption band were measured in the pH range between 7.7 and 4.5. The vibrational band pattern in the Cu-cysteine stretching region is closely related to that of azurin, indicating far-reaching similarities of the coordination geometry of the copper center in both proteins. Significantly lower frequencies, however, are noted for the Cu-histidine stretches, which appear as a closely spaced doublet at ca. 260 cm-1. Lowering the pH to 4.5 leads to an increase of this band splitting with one component shifting down to 247 cm-1. This downshift is attributed to the rupture of a hydrogen bond between one of the histidine ligands and a nearby carboxyl group, which becomes protonated at such a low pH. On the other hand, no major changes in the Cu-cysteine stretching region are noted at pH 4.5, implying that the coordination geometry remains largely unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Azurin↗
PubMed1987

Hydrogen bonding of sulfur ligands in blue copper and iron-sulfur proteins: detection by resonance Raman spectroscopy.

The resonance Raman spectrum of the blue copper protein azurin from Alcaligenes denitrificans exhibits nine vibrational modes between 330 and 460 cm-1, seven of which shift 0.4-3.0 cm-1 to lower energy after incubation of the protein in D2O. These deuterium-dependent shifts have been previously ascribed to exchangeable protons on imidazole ligands [Nestor, L., Larrabee, J. A., Woolery, G., Reinhammar, B., & Spiro, T. G. (1984) Biochemistry 23, 1084] or to exchangeable protons on amide groups which are hydrogen bonded to the cysteine thiolate ligands (a feature common to all blue copper proteins of known structure). In order to distinguish between these two possibilities, a systematic investigation of Fe2S2(Cys)4-containing proteins was undertaken. Extensive hydrogen bonding between sulfur ligands and the polypeptide backbone had been observed in the crystal structure of ferredoxin from Spirulina platensis. The resonance Raman spectrum of this protein is typical of a chloroplast-type ferredoxin and exhibits deuterium-dependent shifts of -0.3 to -0.5 cm-1 in the Fe-S modes at 283, 367, and 394 cm-1 (assigned to the bridging sulfurs) and -0.6 to -0.8 cm-1 in the Fe-S modes at 328 and 341 cm-1 (assigned to the terminal cysteine thiolates). Considerably greater deuterium sensitivity is observed in the Raman spectra of spinach ferredoxin and bovine adrenodoxin, particularly for the symmetric stretching vibration of the Fe2S2 moiety at approximately 390 cm-1. This feature decreases by 0.8 and 1.1 cm-1, respectively, for the two oxidized proteins in D2O and by 1.8 cm-1 for reduced adrenodoxin in D2O.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗
PubMed1977

Study of the triplet state properties of tyrosines and tryptophan in azuring using optically detected magnetic resonance.

Optically detected magnetic resonance (ODMR) signals and phosphorescence spectra were seen of tyrosine in the P. aeruginosa and tryptophanless P. fluorescens azurins and of tryptophan in the former. This confirmed a conclusion from other experiments that the tryptophan of P. aeruginosa cannot effectively quench the singlet energy of both tyrosines. The ODMR signals were all very narrow, additional evidence that the chromophores are buried in the interior of the protein. Accurate values of the zero-field coupling constants D and E lead to a tentative correlation of D values with the red shift of the 0 leads to 0 peak of the phosphorescence spectrum. The environment of tryptophan in P. aeruginosa is the most hydrocarbon like of any tryptophan so far observed. The experiments raise a number of unanswered questions concerning rate processes. The intensities of the 2E transition of tyrosine and the phosphorescence of both tyrosine and tryptophan are substantially reduced when the copper is oxidized. Nevertheless the phsphorescence lifetimes are unaffected. A hole cannot be burned in the ODMR resonances. The homogeneously broadened lines may conceivably be a result of low-temperature proton tunnelling.

Azurin↗
PubMed1975

The copper coordination group in "blue" copper proteins: evidence from resonance Raman spectra.

Tunable dye laser excitation in the intense similar to 600-nm absorption band of azurin, plastocyanin, and ceruloplasmin provides resonance enhanced Raman spectra. They consist of a complex set of bands, at least three or four in number, between 350 and 473 cm-1, which are assignable to Cu-N or Cu-O bond stretching, and a weak band near 270 cm-1, which probably arises from Cu-S stretching. A weak band at 765 cm-1 found in plastocyanin may arise from C-S stretching. Analysis of the Raman intensity pattern, as well as of the nature of the resonant electronic transition, leads to a model of the "blue" copper site involving approximately trigonal-bipyramidal coordination, with a sulfur and two nitrogen ligands in the equatorial plane, and less strongly bound nitrogen or oxygen ligands at axial positions. This arrangement would be well poised for stabilization of Cu(I) upon reduction.

Azurin↗
PubMed2003

Control of metalloprotein reduction potential: compensation phenomena in the reduction thermodynamics of blue copper proteins.

The reduction thermodynamics (Delta H degrees '(rc) and Delta S degrees '(rc)) for native Paracoccus versutus amicyanin, for Alcaligenes faecalis S-6 pseudoazurin, and for the G45P, M64E, and K27C variants of Pseudomonas aeruginosa azurin were measured electrochemically. Comparison with the data available for other native and mutated blue copper proteins indicates that the features of metal coordination and the electrostatic potential due to the protein matrix and the solvent control the reduction enthalpy in a straightforward way. However, the effects on the reduction potential are rather unpredictable owing to the entropic contribution to E degrees ', which is mainly determined by solvent reorganization effects. Analysis of all the Delta H degrees '(rc) and Delta S degrees '(rc) values available for this protein class indicates that enthalpy-entropy compensation occurs in the reduction thermodynamics of wt cupredoxins from different sources, as well as for mutants of the same species. The findings indicate that the reduction enthalpies and entropies for these species are strongly affected by reduction-induced reorganization of solvent molecules within the solvation sphere of the protein. The absence of a perfect enthalpy-entropy compensation is due to the fact that while the differences between reduction entropies are dominated by solvent reorganization effects, those between reduction enthalpies are significantly controlled by intrinsic molecular factors related to the selective stabilization of the reduced form by coordination features of the copper site and electrostatic effects at the interface with the protein matrix.

Alcaligenes↗
PubMed2005

Ligand loop effects on the free energy change of redox and pH-dependent equilibria in cupredoxins probed on amicyanin variants.

In this work, we have determined the thermodynamic parameters of the reduction of four different variants of Thiobacillus versutus amicyanin by electrochemical techniques. In addition, the thermodynamic parameters were determined of the low-pH conformational change involving protonation of the C-terminal histidine ligand and the concomitant dissociation of this histidine from the Cu(I) ion. In these variants, the native C-terminal loop containing the Cys, His, and Met copper ligands has been replaced with the corresponding polypeptide segments of Pseudomonas aeruginosa azurin, Populus nigra plastocyanin, Alcaligenes faecalis S-6 pseudoazurin, and Thiobacillus ferrooxidans rusticyanin. For the reduction reaction, each loop invariably holds an entropic "memory" of the mother protein. The thermodynamics of the low-pH transition vary in a fashion that is species-dependent. When present, the memory effect again shows a large entropic component. In particular, loop elongation tends to favor the formation of the Cu(I)-His bond (hence disfavors His protonation, yielding lower pK(a) values) probably due to an increased flexibility of the loop in the reduced state. Overall, it appears that both reduction and low-pH transition are loop-responsive processes. The spacing between the ligands mostly affects the change in the conformational freedom that accompanies the reaction.

Amino Acid Substitution↗
PubMed1996

Analysis of the paramagnetic copper(II) site of amicyanin by 1H NMR spectroscopy.

Application of the tailored pulse sequences like super-WEFT allows the direct observation of the hyperfine-shifted signals of the paramagnetic Cu(II) forms of blue copper proteins in solution. The signals can be assigned by applying 2D NMR techniques, like EXSY, to solutions containing a mixture of reduced and oxidized species. The Fermi contact shift is separated from the pseudocontact shift on the basis of the known g-tensor anisotropy of the Cu(II) state, allowing the determination of a number of hyperfine-splitting constants between protons on the Cu ligands and the unpaired electron. These results are used to quantify the spin density distribution over the Cu ligands. In amicyanin about 50%-60% of the unpaired electron density is found on the ligands. It appears possible to quantify the Cu-S(Met) interaction on the basis of the NMR results. Application of the technique to the wild type forms of amicyanin and azurin and to two active site mutants of amicyanin (His96Asp and a plastocyanin-amicyanin loop exchange mutant) shows that the Cu-S(Met) interaction parallels the rhombicity and axial distortion of the Cu site.

Amino Acid Sequence↗
PubMed2001

A quantitative description of the ground-state wave function of Cu(A) by X-ray absorption spectroscopy: comparison to plastocyanin and relevance to electron transfer.

To evaluate the importance of the electronic structure of Cu(A) to its electron-transfer (ET) function, a quantitative description of the ground-state wave function of the mixed-valence (MV) binuclear Cu(A) center engineered into Pseudomonas aeruginosa azurin has been developed, using a combination of S K-edge and Cu L-edge X-ray absorption spectroscopies (XAS). Parallel descriptions have been developed for a binuclear thiolate-bridged MV reference model complex ([(L(i)(PrdacoS)Cu)(2)](+)) and a homovalent (II,II) analogue ([L(i)(Pr2tacnS)Cu)(2)](2+), where L(i)(PrdacoS) and L(i)(Pr2tacnS) are macrocyclic ligands with attached thiolates that bridge the Cu ions. Previous studies have qualitatively defined the ground-state wave function of Cu(A) in terms of ligand field effects on the orbital orientation and the presence of a metal--metal bond. The studies presented here provide further evidence for a direct Cu--Cu interaction and, importantly, experimentally quantify the covalency of the ground-state wave function. The experimental results are further supported by DFT calculations. The nature of the ground-state wave function of Cu(A) is compared to that of the well-defined blue copper site in plastocyanin, and the importance of this wave function to the lower reorganization energy and ET function of Cu(A) is discussed. This wave function incorporates anisotropic covalency into the intra- and intermolecular ET pathways in cytochrome c oxidase. Thus, the high covalency of the Cys--Cu bond allows a path through this ligand to become competitive with a shorter His path in the intramolecular ET from Cu(A) to heme a and is particularly important for activating the intermolecular ET path from heme c to Cu(A).

Azurin↗
PubMed2002

Electron-mediating Cu(A) centers in proteins: a comparative high field (1)H ENDOR study.

High field (W-band, 95 GHz) pulsed electron-nuclear double resonance (ENDOR) measurements were carried out on a number of proteins that contain the mixed-valence, binuclear electron-mediating Cu(A) center. These include nitrous oxide reductase (N(2)OR), the recombinant water-soluble fragment of subunit II of Thermus thermophilus cytochrome c oxidase (COX) ba(3) (M160T9), its M160QT0 mutant, where the weak axial methionine ligand has been replaced by a glutamine, and the engineered "purple" azurin (purpAz). The three-dimensional (3-D) structures of these proteins, apart from the mutant, are known. The EPR spectra of all samples showed the presence of a mononuclear Cu(II) impurity with EPR characteristics of a type II copper. At W-band, the g( perpendicular) features of this center and of Cu(A) are well resolved, thus allowing us to obtain a clean Cu(A) ENDOR spectrum. The latter consists of two types of ENDOR signals. The first includes the signals of the four strongly coupled cysteine beta-protons, with isotropic hyperfine couplings, A(iso), in the 7-15 MHz range. The second group consists of weakly coupled protons with a primarily anisotropic character with A(zz) < 3 MHz. Orientation selective ENDOR spectra were collected for N(2)OR, M160QT0, and purpAz, and simulations of the cysteine beta-protons signals provided their isotropic and anisotropic hyperfine interactions. A linear correlation with a negative slope was found between the maximum A(iso) value of the beta-protons and the copper hyperfine interaction. Comparison of the best-fit anisotropic hyperfine parameters with those calculated from dipolar interactions extracted from the available 3-D structures sets limit to the sulfur spin densities. Similarly, the small coupling spectral region was simulated on the basis of the 3-D structures and compared with the experimental spectra. It was found that the width of the powder patterns of the weakly coupled protons recorded at g(perpendicular) is mainly determined by the histidine H(epsilon)(1) protons. Furthermore, the splitting in the outer wings of these powder patterns indicates differences in the positions of the imidazole rings relative to the Cu(2)S(2) core. Comparison of the spectral features of the weakly coupled protons of M160QT0 with those of the other investigated proteins shows that they are very similar to those of purpAz, where the Cu(A) center is the most symmetric, but the copper spin density and the H(epsilon)(1)-Cu distances are somewhat smaller. All proteins show the presence of a proton with a significantly negative A(iso) value which is assigned to an amide proton of one of the cysteines. The simulations of both strongly and weakly coupled protons, along with the known copper hyperfine couplings, were used to estimate and compare the spin density distribution in the various Cu(A) centers. The largest sulfur spin density was found in M160T9, and the lowest was found in purpAz. In addition, using the relation between the A(iso) values of the four cysteine beta-protons and the H-C-S-S dihedral angles, the relative contribution of the hyperconjugation mechanism to A(iso) was determined. The largest contribution was found for M160T9, and the lowest was found for purpAz. Possible correlations between the spin density distribution, structural features, and electron-transfer functionality are finally suggested.

Azurin↗
PubMed2002

Metal-ligand interplay in blue copper proteins studied by 1H NMR spectroscopy: Cu(II)-pseudoazurin and Cu(II)-rusticyanin.

The blue copper proteins (BCPs), pseudoazurin from Achromobacter cycloclastes and rusticyanin from Thiobacillus ferrooxidans, have been investigated by (1)H NMR at a magnetic field of 18.8 T. Hyperfine shifts of the protons belonging to the coordinated ligands have been identified by exchange spectroscopy, including the indirect detection for those resonances that cannot be directly observed (the beta-CH(2) of the Cys ligand, and the NH amide hydrogen bonded to the S(gamma)(Cys) atom). These data reveal that the Cu(II)-Cys interaction in pseudoazurin and rusticyanin is weakened compared to that in classic blue sites (plastocyanin and azurin). This weakening is not induced by a stronger interaction with the axial ligand, as found in stellacyanin, but might be determined by the protein folding around the metal site. The average chemical shift of the beta-CH(2) Cys ligand in all BCPs can be correlated to geometric factors of the metal site (the Cu-S(gamma)(Cys) distance and the angle between the CuN(His)N(His) plane and the Cu-S(gamma)(Cys) vector). It is concluded that the degree of tetragonal distortion is not necessarily related to the strength of the Cu(II)-S(gamma)(Cys) bond. The copper-His interaction is similar in all BCPs, even for the solvent-exposed His ligand. It is proposed that the copper xy magnetic axes in blue sites are determined by subtle geometrical differences, particularly the orientation of the His ligands. Finally, the observed chemical shifts for beta-CH(2) Cys and Ser NH protons in rusticyanin suggest that a less negative charge at the sulfur atom could contribute to the high redox potential (680 mV) of this protein.

Alcaligenes↗
PubMed2003

Spectroscopy and reactivity of a photogenerated tryptophan radical in a structurally defined protein environment.

Near-UV irradiation of structurally characterized [Re(I)(CO)3(1,10-phenanthroline)(Q107H)](W48F/Y72F/H83Q/Y108W)AzM(II) [Az = Pseudomonas aeruginosa azurin, M = Cu, Zn]/[Co(NH3)5Cl]Cl2 produces a tryptophan radical (W108*) with unprecedented kinetic stability. After rapid formation (k = 2.8 x 106 s-1), the radical persists for more than 5 h at room temperature in the folded ReAzM(II) structure. The absorption spectrum of ReAz(W108*)M(II) exhibits maxima at 512 and 536 nm. Oxidation of K4[Mo(CN)8] by ReAz(W108*)Zn(II) places the W108*/W108 reduction potential in the protein above 0.8 V vs NHE.

Azurin↗
PubMed2004

Exploring the electronic and mechanical properties of protein using conducting atomic force microscopy.

In interfacing man-made electronic components with specifically folded biomacromolecules, the perturbative effects of junction structure on any signal generated should be considered. We report herein on the electron-transfer characteristics of the blue copper metalloprotein, azurin, as characterized at a refined level by conducting atomic force microscopy (C-AFM). Specifically, the modulation of current-voltage (I-V) behavior with compressional force has been examined. In the absence of assignable resonant electron tunneling within the confined bias region, from -1 to 1 V, the I-V behavior was analyzed with a modified Simmons formula. To interpret the variation of tunneling barrier height and barrier length obtained by fitting with the modified Simmons formula, an atom packing density model associated with protein mechanical deformation was proposed and simulated by molecular dynamics. The barrier heights determined at the minimum forces necessary for stable electrical contact correlate reasonably well with those estimated from bulk biophysical (electroanalytical and photochemical) experiments previously reported. At higher forces, the tunnel barrier decreases to fall within the range observed with saturated organic systems. Molecular dynamics simulations revealed changes in secondary structure and atomic density of the protein with respect to compression. At low compression, where transport measurements are made, secondary structure is retained, and atomic packing density is observed to increase linearly with force. These predictions, and those made at higher compression, are consistent with both experimentally observed modulations of tunneling barrier height with applied force and the applicability of the atom packing density model of electron tunneling in proteins to molecular-level analyses.

Azurin↗
PubMed2004

Loop-contraction mutagenesis of type 1 copper sites.

The shortest known type 1 copper binding loop (that of amicyanin, Ami) has been introduced into three different cupredoxin beta-barrel scaffolds. All of the loop-contraction variants possess copper centers with authentic type 1 properties and are redox active. The Cu(II) and Co(II) sites experience only small structural alterations upon loop contraction with the largest changes in the azurin variant (AzAmi), which can be ascribed to the removal of a hydrogen bond to the coordinating thiolate sulfur of the Cys ligand. In all cases, loop contraction leads to an increase in the pK(a) of the His ligand found on the loop in the reduced proteins, and in the pseudoazurin (Paz) and plastocyanin (Pc) variants the values are almost identical to that of Ami ( approximately 6.7). Thus, in Paz, Pc, and Ami, the length of this loop tunes the pK(a) of the His ligand. In the AzAmi variant, the pK(a) is 5.5, which is considerably higher than the estimated value for Az (<2), and other controlling factors, along with loop length, are involved. The reduction potentials of the loop-contraction variants are all lower than those of the wild-type proteins by approximately 30-60 mV, and thus this property of a type 1 copper site is fine-tuned by the C-terminal loop. The electron self-exchange rate constant of Paz is significantly diminished by the introduction of a shorter loop. However, in PcAmi only a 2-fold decrease is observed and in AzAmi there is no effect, and thus in these two cupredoxins loop contraction does not significantly influence electron-transfer reactivity. Loop contraction provides an active site environment in all of the cupredoxins which is preferable for Cu(II), whereas previous loop elongation experiments always favored the cuprous site. Thus, the ligand-containing loop plays an important role in tuning the entatic nature of a type 1 copper center.

Azurin↗
PubMed2007

Engineering copper sites in proteins: loops confer native structures and properties to chimeric cupredoxins.

The ligand-containing loops of two copper-binding electron-transfer proteins (cupredoxins) have been swapped. In the azurin (AZ) variant in which the plastocyanin (PC) sequence is introduced (AZPC), the loop adopts a conformation identical to that in PC. The reduction potential of AZPC is raised as compared to AZ and matches that of PC. In the previously published AZAMI variant (AMI = amicyanin), the shorter introduced loop adopts the same conformation as in AMI, and the reduction potential is lowered to equal that of AMI (Yanagisawa, S.; Dennison, C. J. Am. Chem. Soc. 2004, 126, 15711-15719. Li, C.; et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 7258-7263). Thus, the loop structure plays an important role in tuning the reduction potential of a type 1 copper site with contributions from protein dipoles in this region probably the most important feature. The structure of the loop also seems to be a major factor in controlling dissociation and protonation of the C-terminal His ligand, which can act as a switch to regulate electron-transfer reactivity. The PCAZ variant (PC with the AZ loop) possesses an active site, which is different from those of both PC and AZ, and it is assumed that the introduced loop does not adopt a structure as in AZ. This contributes to the observed instability of PCAZ and highlights that loop-scaffold interactions are important for stabilizing the active site of a cupredoxin.

Azurin↗
PubMed2006

Monitoring interfacial bioelectrochemistry using a FRET switch.

Generation of functionally active biomolecular monolayers is important in both analytical science and biophysical analyses. Our ability to monitor the redox-active state of immobilized proteins or enzymes at a molecular level, from which stochastic and surface-induced variations would be apparent, is impeded by comparatively slow electron-transfer kinetics and associated signal:noise difficulties. We demonstrate herein that by covalently tethering an appropriate dye to the copper protein azurin a highly oxidation-state-sensitive FRET process can be established which enables redox switching to be optically monitored at protein levels down to the zeptomolar limit. The surface-potential-induced cycling of emission enables the redox potential of clusters of a few hundred molecules to be determined.

Azurin↗
PubMed2003

Fully automated sequence-specific resonance assignments of hetero- nuclear protein spectra.

Full automation of the analysis of spectra is a prerequisite for high-throughput NMR studies in structural or functional genomics. Sequence-specific assignments often form the major bottleneck. Here, we present a procedure that yields nearly complete backbone and side chain resonance assignments starting from a set of heteronuclear three-dimensional spectra. Neither manual intervention, e.g., to correct lists obtained from peak picking before feeding these to an assignment program, nor protein-specific information, e.g., structures of homologous proteins, were required. By combining two earlier published procedures, AUTOPSY [Koradi et al. (1998) J. Magn. Reson., 135, 288-297] and GARANT [Bartels et al. (1996) J. Biomol. NMR, 7, 207-213], with a new program, PICS, all necessary steps from spectra analyses to sequence-specific assignments were performed fully automatically. Characteristic features of the present approach are a flexible design allowing as input almost any combination of NMR spectra, applicability to side chains, robustness with respect to parameter choices (such as noise levels) and reproducibility. In this study, automated resonance assignments were obtained for the 14 kD blue copper protein azurin from P. aeruginosa using five spectra: HNCACB, HNHA, HCCH-TOCSY, 15N-NOESY-HSQC and 13C-NOESY-HSQC. Peaks from these three-dimensional spectra were filtered and calibrated with the help of two two-dimensional spectra: 15N-HSQC and 13C-HSQC. The rate of incorrect assignments is less than 1.5% for backbone nuclei and about 3.5% when side chain protons are also considered.

Azurin↗