The effect of fluorescent labeling on α-synuclein fibril morphology.

The misfolding and aggregation of a small, natively unfolded protein α-synuclein (α-syn) is presumably an important factor in the development of Parkinson's disease. However, the mechanism of α-syn aggregation into amyloid fibrils and their morphology are not well understood. To elucidate the aggregation kinetics and the morphology of aggregates by the use of fluorescent techniques the protein needs to be suitably labeled. In this study, using atomic force microscopy, we demonstrate a significant effect of fluorescent labels on the α-syn fibrillization process. We studied in detail the morphology of α-syn aggregates as a function of the composition of mixtures of labeled and wild type (WT) α-syn in solution using different types of fluorescent dyes. Although the overall charge of the fluorophores we used and their chemical structure varied significantly, the morphology of α-syn fibrils changed in a similar way in all cases. The increase in the fraction of labeled α-syn in solution led to shortening of the fibrils as compared to those from WT-only α-syn, whereas the height of the fibrils remained mainly unaffected. The twisted fibril morphology observed in the WT and A140C α-syn mutant completely disappeared when the A140C α-syn mutant was 100% fluorescently labeled.

Redox cycling and kinetic analysis of single molecules of solution-phase nitrite reductase.

Single-molecule measurements are a valuable tool for revealing details of enzyme mechanisms by enabling observation of unsynchronized behavior. However, this approach often requires immobilizing the enzyme on a substrate, a process which may alter enzyme behavior. We apply a microfluidic trapping device to allow, for the first time, prolonged solution-phase measurement of single enzymes in solution. Individual redox events are observed for single molecules of a blue nitrite reductase and are used to extract the microscopic kinetic parameters of the proposed catalytic cycle. Changes in parameters as a function of substrate concentration are consistent with a random sequential substrate binding mechanism.

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.

The solution structure of the guanine nucleotide exchange domain of human elongation factor 1beta reveals a striking resemblance to that of EF-Ts from Escherichia coli.

BACKGROUND: In eukaryotic protein synthesis, the multi-subunit elongation factor 1 (EF-1) plays an important role in ensuring the fidelity and regulating the rate of translation. EF-1alpha, which transports the aminoacyl tRNA to the ribosome, is a member of the G-protein superfamily. EF-1beta regulates the activity of EF-1alpha by catalyzing the exchange of GDP for GTP and thereby regenerating the active form of EF-1alpha. The structure of the bacterial analog of EF-1alpha, EF-Tu has been solved in complex with its GDP exchange factor, EF-Ts. These structures indicate a mechanism for GDP-GTP exchange in prokaryotes. Although there is good sequence conservation between EF-1alpha and EF-Tu, there is essentially no sequence similarity between EF-1beta and EF-Ts. We wished to explore whether the prokaryotic exchange mechanism could shed any light on the mechanism of eukaryotic translation elongation. RESULTS: Here, we report the structure of the guanine-nucleotide exchange factor (GEF) domain of human EF-1beta (hEF-1beta, residues 135-224); hEF-1beta[135-224], determined by nuclear magnetic resonance spectroscopy. Sequence conservation analysis of the GEF domains of EF-1 subunits beta and delta from widely divergent organisms indicates that the most highly conserved residues are in two loop regions. Intriguingly, hEF-1beta[135-224] shares structural homology with the GEF domain of EF-Ts despite their different primary sequences. CONCLUSIONS: On the basis of both the structural homology between EF-Ts and hEF-1beta[135-224] and the sequence conservation analysis, we propose that the mechanism of guanine-nucleotide exchange in protein synthesis has been conserved in prokaryotes and eukaryotes. In particular, Tyr181 of hEF-1beta[135-224] appears to be analogous to Phe81 of Escherichia coli EF-Ts.

EXAFS analysis of the pH dependence of the blue-copper site in amicyanin from Thiobacillus versutus.

The room temperature Cu K-edge EXAFS (extended X-ray absorption fine structure) spectrum of reduced and oxidized amicyanin, the blue copper protein from Thiobacillus versutus, was measured at low and high pH. The data interpretation was partly based on independent NMR evidence for the occurrence of a ligand histidine protonation at low pH (pKa = 6.9) in the reduced protein. In the oxidized protein two nitrogen-donors (from two histidines; Cu-N distances 1.95-2.01 A and 1.86-1.89 A) and a sulfur-donor (from a cysteine; Cu-S distance 2.11-2.13 A) were identified and the coordination appears independent of pH. Upon reduction at high pH the Cu-S bond and one of the Cu-N bonds lengthen slightly (from 2.11 to 2.19 A and from 2.01 to 2.18 A, respectively). Upon lowering of the pH one of the N-donors of the Cu in reduced amicyanin disappears from the Cu EXAFS and a second S-donor (from a methionine) becomes visible at 2.41 A from the Cu. The Debye-Waller factors are compatible with a Cu-N vibrational stretch frequency in the range of 150-250 cm-1 and one greater than 285 cm-1, and a Cu-S vibrational stretch frequency of about 150 cm-1 (Cu-Smet; reduced amicyanin at low pH) and one in the range of 230-800 cm-1 (Cu-Scys).

Purification and characterization of a non-reconstitutable azurin, obtained by heterologous expression of the Pseudomonas aeruginosa azu gene in Escherichia coli.

The azurin-encoding azu gene from Pseudomonas aeruginosa was cloned and expressed in Escherichia coli. A purification procedure was developed to isolate the azurin obtained from the E. coli cells. No differences were observed between azurins isolated from P. aeruginosa and E. coli. A non-reconstitutable azurin-like protein, azurin*, with a spectral ratio (A625/A280) less than 0.01 could be separated from holo-azurin with a spectral ratio of 0.58 (+/- 0.01). The properties of azurin* were examined by electrophoretic (SDS-PAGE and IEF) and spectroscopic (UV/vis, 1H-NMR, static and dynamic fluorescence) techniques, and compared to the properties of holo-azurin and apo-azurin. Azurin* resembles apo-azurin (same pKa* values of His-35 and His-117, same fluorescence characteristics). However, it has lost the ability to bind Cu-ions. It is tentatively concluded that azurin* is a chemically modified form of azurin, the modification possibly being due to oxidation of the ligand residue Cys-112 or the formation of a chemical bond between the ligand residues Cys-112 and His-117. In agreement with previous results from Hutnik and Szabo (Biochemistry (1989) 28, 3923-3934), fluorescence experiments show that the heterogeneous fluorescence decay observed for holo-azurin is not due to the presence of azurin*, but most likely originates from conformational heterogeneity of the holo-azurin.

The pH and redox-state dependence of the copper site in azurin from Pseudomonas aeruginosa as studied by EXAFS.

The EXAFS of the K-edge of copper in azurin from Pseudomonas aeruginosa has been measured in solutions of the oxidized and reduced protein, at both low and high pH. Model compounds of known molecular structure, exhibiting Cu-N and Cu-S bonds of varying length, were studied as well. The major shell of the high-pH oxidized azurin EXAFS contains contributions of two N(His) at 1.95 +/- 0.03 A, and one S(Cys) at 2.23 +/- 0.03 A. Some minor contributions from the carbon atoms of the histidine residues and the distal sulfur atom are observed in the 3-4 A region. Upon reduction a decrease is seen in amplitude of the main peak in the Fourier transform, due to a lengthening of one of the Cu-N(His) bonds (2.05 +/- 0.03 A), and a shortening of the other (1.89 +/- 0.03 A), both by approx. 0.1 A. Indications for a Cu-S(Met) bond are found in the reduced azurin data (2.70 +/- 0.05 A). However, in the oxidized protein, this bond could not be determined unambiguously, in line with results of a model compound featuring weak Cu-thioether coordination. The effect of pH is only slight for both the oxidized and the reduced protein, and no significant changes in bond lengths are found upon a change of pH from 4.1 to 9.1. The relevance of these findings for the interpretation of the existing data on the redox activity of the protein is discussed.

Cloning and sequence analysis of the gene encoding Methylophilus methylotrophus cytochrome c", a unique protein with a perpendicular orientation of the histidinyl ligands.

Cytochrome c" from Methylophilus methylotrophus is an unusual monohaem protein that undergoes a major redox-linked spin-state transition: one of the two axial histidines bound to the iron in the oxidised form is detached upon reduction and a proton is taken up. A 3.5-kb DNA fragment, containing the gene encoding cytochrome c" (cycA), has been cloned and sequenced. The cytochrome c" gene codes for a pre-protein with a typical prokaryotic 20-residue signal sequence, suggesting that the protein is synthesised as a precursor which is processed during its secretion into the periplasm. The C-terminus of cytochrome c" has homology with the corresponding region of an oxygen-binding haem protein (SHP) from phototrophically grown Rhodobacter sphaeroides. SHP is similar in size and in the location of its haem-binding site. Immediately downstream from cytochrome c" a second open reading frame (ORF) codes for a 23-kDa protein with similarity to the cytochrome b-type subunit of Ni-Fe hydrogenase. The possibility of coordinated expression of cycA and this ORF is discussed.

Electrostatic effects of charge perturbations introduced by metal oxidation in proteins. A theoretical analysis.

A macroscopic dielectric model for the interactions between charges in proteins is used to calculate the changes in His residue pKa values induced in azurin by oxidation of the copper. The calculated results agree with nuclear magnetic resonance experiments to within the uncertainty associated with the measurements. It is found that a large apparent dielectric constant can describe the interaction between two protein groups, even if the shortest path between them is through the protein, which is assumed to have a low dielectric constant.

1H nuclear magnetic resonance study of the protonation behaviour of the histidine residues and the electron self-exchange reaction of azurin from Alcaligenes denitrificans.

The proton nuclear magnetic resonance spectrum of azurin from Alcaligenes denitrificans at pH 6.0 and 309 K is reported. Proton signals from all methionine and histidine residues (among them the copper ligands) have been assigned. The data have been used to study the pH behaviour of His35 and to establish the electron self-exchange rate of the protein. His35 appears to be protonated at pH less than 4.5, possibly after rupture of a salt bridge. No effects of this protonation on the tertiary structure around the copper site are observed, however, contrary to the case of Pseudomonas aeruginosa azurin. The electron self-exchange rate amounts to 4 x 10(5) M-1 S-1 at pH 6.7 and 297 K. The data support the conclusion that the electron self-exchange takes place by way of the hydrophobic surface patch around His117, and that His35 is not involved in this reaction. Oxidation of azurin increases the acidity of the freely titrating His32 and His83 by 0.07 and 0.25 pKa units, respectively. The data can be used to test the theory of electrostatic interactions in proteins. The optical extinction coefficient at 625 nm was experimentally determined and amounts to 4.8(+/- 0.1) x 10(3) M-1 cm-1.

X-ray analysis and spectroscopic characterization of M121Q azurin. A copper site model for stellacyanin.

The dependence of the properties of the azurin blue copper site on the nature of the axial ligand at position 121 was tested by site-directed mutagenesis. This residue was substituted for a glutamine, the purported fourth copper ligand in the related protein stellacyanin. M121Q azurin was isolated and purified from Escherichia coli and characterized by spectroscopic methods. The mutant copper site has the ultra-violet-vis and electron paramagnetic resonance (EPR) characteristics of a type I site, but the spectroscopic details differ significantly from wild-type (wt) azurin. The X and S-band EPR spectra of M121Q azurin can be well stimulated with the parameters for stellacyanin, indicating that the copper sites of both proteins in the oxidized state are similar. The midpoint potential of M121Q is 263 mV, 25 mV lower than for wt azurin. The reactivity of the mutant was probed by measuring the electron self exchange rate by nuclear magnetic resonance spectroscopy. The rate was 8 x 10(3) mol-1 s-1, almost two orders of magnitude lower than the value for wt azurin (5 x 10(5) mol-1 s-1). Detailed structural information on the M121Q Cu(II)-site was obtained by X-ray analysis of M121Q azurin crystals at 1.9 A resolution. The histidine and cysteine copper ligand distances and angles in the equatorial plane around the copper are very similar to the wt protein. Gln121 is co-ordinated in a monodentate fashion via its side-chain oxygen atom at a distance of 2.26 A. The distance between copper and the carbonyl group of Gly45 is increased from 3.13 A (wt) to 3.37 A resulting in a distorted tetrahedral N2SO copper co-ordination. The possible significance of these results for the structure of the copper site of stellacyanin, the only small blue copper protein lacking a methionine ligand, is discussed. Conformational changes with respect to the wt azurin are seen in some of the connecting loops between secondary structure elements, in the mutation site and in the beta-strand 2a. The side-chains involved in the hydrophobic patch surrounding His117 are subject to large changes in their conformations. In contrast to wt azurin, the copper site in M121Q azurin undergoes significant structural changes on reduction.(ABSTRACT TRUNCATED AT 400 WORDS)

Crystal structure analysis of oxidized Pseudomonas aeruginosa azurin at pH 5.5 and pH 9.0. A pH-induced conformational transition involves a peptide bond flip.

The X-ray crystal structure of recombinant wild-type azurin from Pseudomonas aeruginosa was determined by difference Fourier techniques using phases derived from the structure of the mutant His35Leu. Two data sets were collected from a single crystal of oxidized azurin soaked in mother liquor buffered at pH 5.5 and pH 9.0, respectively. Both data sets extend to 1.93 A resolution. The two pH forms were refined independently to crystallographic R-factors of 17.6% (pH 5.5) and 17.5% (pH 9.0). The conformational transition previously attributed to the protonation/deprotonation of residue His35 (pKa(red) = 7.3, pKa(ox) = 6.2), which lies in a crevice of the protein close to the copper binding site, involves a concomitant Pro36-Gly37 main-chain peptide bond flip. At the lower pH, the protonated imidazole N delta 1 of His35 forms a strong hydrogen bond with the carbonyl oxygen from Pro36, while at alkaline pH the deprotonated N delta 1 acts as an acceptor of a weak hydrogen bond from HN Gly37. The structure of the remainder of the azurin molecule, including the copper binding site, is not significantly affected by this transition.

X-ray crystal structure of the two site-specific mutants His35Gln and His35Leu of azurin from Pseudomonas aeruginosa.

The three-dimensional structures of two site-specific mutants of the blue copper protein azurin from Pseudomonas aeruginosa have been solved by a combination of isomorphous replacement and Patterson search techniques, and refined by energy-restrained least-squares methods. The mutations introduced by recombinant DNA techniques involve residue His35, which was exchanged for glutamine and leucine, to probe for its suggested role in electron transfer. The two mutants, His35Gln (H35Q) and His35Leu (H35L), crystallize non-isomorphously in the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 109.74 A, b = 99.15 A, c = 47.82 A for H35Q, a = 57.82 A, b = 81.06 A, c = 110.03 A for H35L. In each crystal form, there are four molecules in the asymmetric unit. They are arranged as a dimer of dimers in the H35Q case and are distorted from ideal C2 symmetry in H35L. The final crystallographic R-value is 16.3% for 20.747 reflections to a resolution of 2.1 A for H35Q and 17.0% for 32,548 reflections to 1.9 A for H35L. The crystal structures reported here represent the first crystallographically refined structures for azurin from P. aeruginosa. The structure is very similar to that of azurin from Alcaligenes denitrificans. The copper atom is located about 7 A below a hydrophobic surface region and is ligated by five donor groups in a distorted trigonal bipyramidal fashion. The implications for electron transfer properties of the protein are discussed in terms of the mutation site and the packing of the molecules within the tetramer.

The structural role of the copper-coordinating and surface-exposed histidine residue in the blue copper protein azurin.

Copper K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy and (15)N NMR relaxation studies were performed on samples of a variant azurin in which the surface-exposed histidine ligand of the copper atom (His117) has been replaced by glycine. The experiments were performed to probe the structure of the active site and the protein dynamics. The cavity in the protein structure created by the His-->Gly replacement could be filled by external ligands, which can either restore the spectroscopic properties of the original type-1 copper site or create a new type-2 copper site. The binding of external ligands occurs only when the copper atom is in its oxidised state. In the reduced form, the binding is abolished. From the EXAFS experiments, it is concluded that for the oxidised type-1 copper sites the protein plus external ligand (L) provide an NSS*L donor set deriving from His46, Cys112, Met121 and the external ligand. The type-2 copper site features an S(N/O)(3) donor set in which the S-donor derives from Cys112, one N-donor from His46 and the remaining two N or O donors from one or more external ligands. Upon reduction of the type-1 as well as the type-2 site, the external ligand drops out of the copper site and the coordination reduces to 3-fold with an SS*N donor set deriving from His46, Cys112 and Met121. The Cu-S(delta)(Met) distance is reduced from about 3.2 to 2.3 A. Analysis of the NMR data shows that the hydrophobic patch around His117 has gained fluxionality when compared to wild-type azurin, which may explain why the His117Gly variant is able to accommodate a variety of external ligands of different sizes and with different chelating properties. On the other hand, the structure and dynamics of the beta-sandwich, which comprises the main body of the protein, is only slightly affected by the mutation. The unusually high reduction potential of the His117Gly azurin is discussed in light of the present results.

X-ray crystal structure of the two site-specific mutants Ile7Ser and Phe110Ser of azurin from Pseudomonas aeruginosa.

The blue copper protein azurin from Pseudomonas aeruginosa contains a single Trp residue that is believed to be involved in the inducible intramolecular electron transfer from a disulphide group to the copper centre. This residue shows in fluorescence spectra the highest energy emission of tryptophan-containing compounds at room temperature, which is explained by its rigid and highly hydrophobic environment. In order to investigate the role of the Trp residue in electron transfer and the influence of its environment, two mutations (17S and F110S) were introduced that were thought to increase the polarity and the mobility in its environment. The crystal structures of these mutants were solved at 2.2 A and 2.3 A resolution, respectively. These provide a structural basis for the changes observed in fluorescence spectra compared with the wild-type protein. We conclude from our results that these changes are not caused by a change in the dynamics of the Trp residue itself, but exclusively by an increased effective dielectric constant of the microenvironment of Trp48 and by changes in mobility of the mutated residues.

Solution structure of the type 1 blue copper protein amicyanin from Thiobacillus versutus.

A three-dimensional solution structure of amicyanin from Thiobacillus versutus has been determined by distance geometry and restrained molecular dynamics. A total of 984 experimentally derived constraints were used for the final refinement (881 distance constraints and 103 dihedral angle constraints). Stereospecific assignments were made for 17 prochiral beta-methylene protons (33%) and the methyl groups of eight valine residues. Fourteen structures were selected to represent the solution structure. They show an average pairwise backbone root-mean-square deviation of 1.19 A. The overall structure can be described as a beta-sandwich, built up of nine beta-strands. The copper atom is located between three loops on one end of the molecule. Two of these loops contribute the copper ligands. His54 is on the loop between beta-strands 4 and 5. The other three ligands, Cys93, His96 and Met99, are located evenly spaced on the loop between beta-strands 8 and 9. This loop is folded in two consecutive type 1 turns with His96 as the donor and acceptor of the NHi-CO(i-3) hydrogen bonds. The folding is reminiscent of the general cupredoxin fold. Considerably different are the large 21 residue N-terminal extension, that is unique to amicyanin and forms an extra beta-strand (strand 1), and the region between beta-strands 5 and 7. The partly surface-exposed copper ligand His96 is surrounded by a hydrophobic patch consisting of seven residues.

Crystal structure analysis and refinement at 2.15 A resolution of amicyanin, a type I blue copper protein, from Thiobacillus versutus.

The crystal structure of the type I blue copper protein amicyanin from Thiobacillus versutus has been determined by Patterson search techniques on the basis of the molecular model of amicyanin from Paracoccus denitrificans, and refined by energy-restrained least-squares methods. Amicyanin crystallizes in the trigonal space group P3(2) with unit cell dimensions of a = b = 87.40 A, c = 38.20 A. The asymmetric unit is composed of three independent molecules centred on the crystallographic 3(2) axes. The final R-value is 17.4% for 15,984 reflections to a resolution of 2.15 A. The polypeptide fold in amicyanin is based on the beta-sandwich structure commonly found in blue copper proteins. Nine beta strands are folded into two twisted beta-sheets that pack together with a filling of non-polar residues between them. The geometry of the copper site is similar to that of plastocyanin. There are four ligands, arranged approximately as a distorted tetrahedron, to the copper atom: His54, Cys93, His96 and Met99. One of the copper ligands, His96, is exposed to the surface and lies in the centre of a cluster of seven hydrophobic residues.

Crystal structures of modified apo-His117Gly and apo-His46Gly mutants of Pseudomonas aeruginosa azurin.

The X-ray crystal structures of two metal ligand mutants of azurin from Pseudomonas aeruginosa have been solved. In both mutants (His117Gly and His46Gly azurin) one of the copper coordinating histidine residues is replaced by a glycine, creating an empty space in the coordination sphere of the copper ion. The crystal structure of His117Gly azurin at 2.4 A resolution showed that this mutant had undergone partial oxidation at the disulfide bridge between Cys3 and Cys26 and full oxidation at the copper ligand Cys112. There is no copper present in the crystallized form and the bulky group of the oxidized cysteine at position 112 causes large structural rearrangements in the protein structure, especially in the loops connecting the beta-sheets. In the structure of the wild-type holo-azurin from P. aeruginosa the hydrophobic patch is important for the packing of the azurin molecules into dimers which then arrange into tetramers. The completely different packing of the apo-His117Gly mutant can be explained by the disruption of the hydrophobic patch area by the mutation-induced main-chain conformational change of residues 112 to 115. The structure of apo-His46Gly azurin at 2.5 A resolution is the same as the wild-type structure except for the immediate environment at the site of the mutation. In the His46Gly structure water molecules are found at positions that in the wild-type structure are occupied by the imidazole ring of His46 and the copper ion. The imidazole ring of His117 is shifted by about 1 A towards the surface of the protein, similar to that observed for 50% of the molecules in the wild-type apo-azurin structure. This shift causes a slight rearrangement of the monomers within the tetramer such that one local dyad becomes a crystallographic dyad parallel to the c-axis. This leads to a change in the space group from P2(1)2(1)2(1) to P2(1)2(1)2.

NMR assignments and relaxation studies of Thiobacillus versutus ferrocytochrome c-550 indicate the presence of a highly mobile 13-residues long C-terminal tail.

Cytochrome c-550 of Thiobacillus versutus functions as an electron transfer protein in a chain of redox proteins that enables T. versutus to grow on methylamine. It is a single-heme protein of 134 residues, related to mitochondrial cytochrome c. Cytochrome c-550, as well as several other bacterial c2-type cytochromes, contain a C-terminal extension of 13-16 amino acids of unknown function, compared to mitochondrial cytochrome c. NMR experiments were performed to obtain structural and dynamic information on the protein in solution. For this purpose, T. versutus cytochrome c-550 was labeled with 15N and 13C using 13C-methanol grown Paracoccus denitrificans as a host for heterologous expression. NMR assignments were obtained for the 1H, 15N, and 13C nuclei in the backbone and the beta-positions of the protein and the secondary structure was determined. 15N-relaxation studies were performed to characterize the dynamic properties of the protein. The results indicate that the main part of T. versutus ferrocytochrome c-550 exists in solution as a rigid, well-ordered molecule with a secondary structure that is very similar to that of P. denitrificans cytochrome c-550, as observed in crystals. The C-terminal extension, however, is unstructured and highly mobile. The possible origin and function of the extension are discussed.

Probing the structure and mobility of Pseudomonas aeruginosa azurin by circular dichroism and dynamic fluorescence anisotropy.

The UV dynamic fluorescence and CD of several Pseudomonas aeruginosa azurins bearing single amino acid mutation have been studied. Two classes of mutants were examined. In the first class, two hydrophobic residues in the core of the protein, Ile 7 and Phe 110, nearest to the azurin single tryptophan Trp 48, were substituted by a serine (mutants 17S and F110S). In the second class, two residues in the outer sphere of the copper ligand field were changed, obtaining the following mutants: M44K, H35F, H35L, and H35Q. All these proteins showed two fluorescence lifetimes in the copper-containing form, but only one in the copper-free form. The lifetime of the latter derivatives was different from either those of the metal-bound samples, definitely ruling out the presence of apo-like species in the holo protein. Copper-free 17S and F110S showed a more complex fluorescence decay profile requiring a distribution of lifetimes rather than a single lifetime. Holo F110S was also better fitted, in the limit of confidence, with two distributions rather than a pair of lifetimes. Time-resolved anisotropy of these two mutants as well as of wild-type (wt) protein showed two components (rotational times for wt < or = 200 ps and 7 ns, respectively). These components were not affected significantly by copper removal in the case of wt protein. Instead, the short rotational component of the mutants dropped dramatically to values near zero, indicating a much greater mobility of the tryptophanyl residue in the mutant apo azurins. These data were supported by CD measurements showing a small effect of the copper presence in the region below 250 nm, i.e., in the secondary structure, but almost a collapse of the aromatic asymmetry at 270-295 nm related to a relaxation of the structural constraint around the tryptophan. Altogether these data show that copper does not play a structural role in wt azurin, whereas it is crucial in the stabilization of 17S and F110S mutants. Furthermore, although the metal site geometry is rigidly kept in wt apo-azurin, it regains the native form only in the presence of the metal in the "core" mutants. This finding is important for the theory of entatic states in metalloproteins (Williams RJP, 1995, Eur J Biochem 234:363-381).