'It can't be zero!' Difficulties in completing patient global assessment in rheumatoid arthritis: a mixed methods study.

OBJECTIVES: Patient global assessment (PGA) is purported to add the patient's perspective in the composite measures of RA. However, PGA is not standardized and it is not known whether patients' interpretation of the measure is consistent with its intended purpose. This study aimed to explore difficulties experienced by patients with RA in completing PGA, and to assess the impact of a structured explanation in improving its validity and reliability. METHODS: This was a mixed methods study, using interviews, focus groups and PGA data. During interviews, patients (convenience sample, n = 33) completed three often-used PGA formulations. Then a nurse provided structured explanation about what PGA is and why it is used. After further discussion, patients completed one PGA version again. Interviews were recorded, transcribed and analysed using inductive thematic analysis. We compared PGA scores pre- and post-explanation (Wilcoxon signed-ranks) and the proportion of patients achieving RA remission with PGA ⩽1 (McNemar's tests). RESULTS: Three themes emerged: understanding the meaning of PGA, the purpose of PGA and measurement difficulties. The difficulties caused systematic errors in PGA completion such as marking higher when feeling well, marking near the centre or away from zero. The structured explanation was helpful. Following the explanation, the median PGA score decreased from 3.0 to 2.1 cm, and the proportion of non-remission solely due to PGA >1 from 52% to 41%; none of these changes was statistically significant. CONCLUSION: Many patients have difficulties in completing PGA. Standardization of PGA and a structured explanation may improve its clarity, validity and reliability.

Insights into the Structures of Superoxide Reductases from the Symbionts Ignicoccus hospitalis and Nanoarchaeum equitans.

Superoxide reductases (SORs) are enzymes that detoxify the superoxide anion through its reduction to hydrogen peroxide and exist in both prokaryotes and eukaryotes. The substrate is transformed at an iron catalytic center, pentacoordinated in the ferrous state by four histidines and one cysteine. SORs have a highly conserved motif, (E)(K)HxP-, in which the glutamate is associated with a redox-driven structural change, completing the octahedral coordination of the iron in the ferric state, whereas the lysine may be responsible for stabilization and donation of a proton to catalytic intermediates. We aimed to understand at the structural level the role of these two residues, by determining the X-ray structures of the SORs from the hyperthermophilic archaea Ignicoccus hospitalis and Nanoarchaeum equitans that lack the quasi-conserved lysine and glutamate, respectively, but have catalytic rate constants similar to those of the canonical enzymes, as we previously demonstrated. Furthermore, we have determined the crystal structure of the E23A mutant of I. hospitalis SOR, which mimics several enzymes that lack both residues. The structures revealed distinct structural arrangements of the catalytic center that simulate several catalytic cycle intermediates, namely, the reduced and the oxidized forms, and the glutamate-free and deprotonated ferric forms. Moreover, the structure of the I. hospitalis SOR provides evidence for the presence of an alternative lysine close to the iron center in the reduced state that may be a functional substitute for the "canonical" lysine.

Superoxide reduction by a superoxide reductase lacking the highly conserved lysine residue.

Superoxide reductases (SORs) are the most recently identified superoxide detoxification systems, being found in microorganisms from the three domains of life. These enzymes are characterized by a catalytic mononuclear iron site, with one cysteine and four histidine ligands of the ferrous active form. A lysine residue in the -EKHVP- motif, located close to the active site, has been considered to be essential for the enzyme function, by contributing to the positive surface patch that attracts the superoxide anion and by controlling the chemistry of the catalytic mechanism through a hydrogen bond network. However, we show here that this residue is substituted by non-equivalent amino acids in several putative SORs from Archaea and unicellular Eukarya. In this work, we focus on mechanistic and spectroscopic studies of one of these less common enzymes, the SOR from the hyperthermophilic Crenarchaeon Ignicoccus hospitalis. We employ pulse radiolysis fast kinetics and spectroscopic approaches to study the wild-type enzyme (-E23T24HVP-), and two mutants, T24K and E23A, the later mimicking enzymes lacking both the lysine and glutamate (a ferric ion ligand) of the motif. The efficiency of the wild-type protein and mutants in reducing superoxide is comparable to other SORs, revealing the robustness of these enzymes to single mutations.

Thermofluor-based optimization strategy for the stabilization and crystallization of Campylobacter jejuni desulforubrerythrin.

Desulforubrerythrin from Campylobacter jejuni has recently been biochemical and spectroscopically characterized. It is a member of the rubrerythrin family, and it is composed of three structural domains: the N-terminal desulforedoxin domain with a non-heme iron center, followed by a four-helix bundle domain harboring a binuclear iron center and finally a C-terminal rubredoxin domain. To date, this is the first example of a protein presenting this kind of structural domain organization, and therefore the determination of its crystal structure may unveil unexpected structural features. Several attempts were made in order to obtain protein crystals, but always without success. As part of our strategy the thermofluor method was used to increase protein stability and its propensity to crystallize. This approach has been recently used to optimize protein buffer formulation, thus yielding more stable and homogenous protein samples. Thermofluor has also been used to identify cofactors/ligands or small molecules that may help stabilize native protein states. A successful thermofluor approach was used to select a pH buffer condition that allowed the crystallization of Campylobacter jejuni desulforubrerythrin, by screening both buffer pH and salt concentration. A buffer formulation was obtained which increased the protein melting temperature by 7°C relatively to the initial purification buffer. Desulforubrerythrin was seen to be stabilized by lower pH and high salt concentration, and was dialyzed into the new selected buffer, 100mM MES pH 6.2, 500mM NaCl. This stability study was complemented with a second thermofluor assay in which different additives were screened. A crystallization screening was carried out and protein crystals were rapidly obtained in one condition. Protein crystal optimization was done using the same additive screening. Interestingly, a correlation between the stability studies and crystallization experiments using the additive screening could be established. The work presented here shows an elegant example where thermofluor was shown to be a key biophysical method that allowed the identification of an improved buffer formulation and the applicability of this technique to increase the propensity of a protein to crystallize is discussed.

Desulforubrerythrin from Campylobacter jejuni, a novel multidomain protein.

A novel multidomain metalloprotein from Campylobacter jejuni was overexpressed in Escherichia coli, purified, and extensively characterized. This protein is isolated as a homotetramer of 24-kDa monomers. According to the amino acid sequence, each monomer was predicted to contain three structural domains: an N-terminal desulforedoxin-like domain, followed by a four-helix bundle domain harboring a non-sulfur µ-oxo diiron center, and a rubredoxin-like domain at the C-terminus. The three predicted iron sites were shown to be present and were studied by a combination of UV-vis, EPR, and resonance Raman spectroscopies, which allowed the determination of the electronic and redox properties of each site. The protein contains two FeCys(4) centers with reduction potentials of +240 mV (desulforedoxin-like center) and +185 mV (rubredoxin-like center). These centers are in the high-spin configuration in the as-isolated ferric form. The protein further accommodates a µ-oxo-bridged diiron site with reduction potentials of +270 and +235 mV for the two sequential redox transitions. The protein is rapidly reoxidized by hydrogen peroxide and has a significant NADH-linked hydrogen peroxide reductase activity of 1.8 µmol H(2)O(2) min(-1) mg(-1). Owing to its building blocks and its homology to the rubrerythrin family, the protein is named desulforubrerythrin. It represents a novel example of the large diversity of the organization of domains exhibited by this enzyme family.

Superoxide reductase from Nanoarchaeum equitans: expression, purification, crystallization and preliminary X-ray crystallographic analysis.

Superoxide reductases (SORs) are the most recent oxygen-detoxification system to be identified in anaerobic and microaerobic bacteria and archaea. SORs are metalloproteins that are characterized by their possession of a catalytic nonhaem iron centre in the ferrous form coordinated by four histidine ligands and one cysteine ligand. Ignicoccus hospitalis, a hyperthermophilic crenarchaeon, is the only organism known to date to serve as a host for Nanoarchaeum equitans, a nanosized hyperthermophilic archaeon isolated from a submarine hot vent which completely depends on the presence of and contact with I. hospitalis cells for growth to occur. Similarly to I. hospitalis, N. equitans has a neelaredoxin (a 1Fe-type SOR) that keeps toxic oxygen species under control, catalysing the one-electron reduction of superoxide to hydrogen peroxide. Blue crystals of recombinant N. equitans SOR in the oxidized form (12.7 kDa, 109 residues) were obtained using polyethylene glycol (PEG 2000 MME) as precipitant. These crystals diffracted to 1.9 Å resolution at 100 K and belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 51.88, b = 82.01, c = 91.30 Å. Cell-content analysis suggested the presence of four monomers in the asymmetric unit. The Matthews coefficient (V(M)) was determined to be 1.9 Å(3) Da(-1), corresponding to an estimated solvent content of 36%. Self-rotation function and native Patterson calculations suggested a tetramer with 222 point-group symmetry, similar to other 1Fe-SORs. The three-dimensional structure will be determined by the molecular-replacement method.

Cloning, purification, crystallization and X-ray crystallographic analysis of Ignicoccus hospitalis neelaredoxin.

Superoxide reductases (SORs) are metalloproteins which constitute the most recently identified oxygen-detoxification system in anaerobic and microaerobic bacteria and archaea. SORs are involved in scavenging superoxide radicals from the cell by catalyzing the reduction of superoxide ({\rm O}_{2};{\bullet -}) to hydrogen peroxide and are characterized by a catalytic nonhaem iron centre coordinated by four histidine ligands and one cysteine ligand. Ignicoccus hospitalis, a hyperthermophilic crenarchaeon, is known to have a neelaredoxin-type SOR that keeps toxic oxygen species levels under control. Blue crystals of recombinant I. hospitalis oxidized neelaredoxin (14.1 kDa, 124 residues) were obtained. These crystals diffracted to 2.4 A resolution in-house at room temperature and belonged to the hexagonal space group P6(2)22 or P6(4)22, with unit-cell parameters a = b = 108, c = 64 A. Cell-content analysis indicated the presence of one monomer in the asymmetric unit.

Resonance Raman study of the superoxide reductase from Archaeoglobus fulgidus, E12 mutants and a 'natural variant'.

The resonance Raman (RR) spectra of the oxidized wild-type Archaeoglobus fuglidus 1Fe-Superoxide reductase (SOR), E12V and E12Q mutants were studied at different pH conditions upon excitation in resonance with the pH-dependent charge transfer transition to the ferric iron. The wild-type SOR from Nanoarchaeum equitans that lacks the highly conserved glutamate residue was investigated as a 'natural variant'. No substantial differences were observed in the RR spectra of the active sites of the A. fulgidus proteins. Based on the component analysis in the metal-ligand stretching region the modes involving the Fe-S(Cys) stretching coordinates have been identified. The frequencies of these modes reflect the electronic properties of the Fe-S bond which are related to the catalytic activity of SORs, including reduction of superoxide and product dissociation. Moreover, hydroxide binding to the E12 mutant proteins was demonstrated at high pH. It was further observed that the ferric active site of all three SORs from A. fulgidus senses the presence of phosphate, which possibly replaces the hydroxide at high pH.