Antioxidant and antibacterial activities, interfacial and emulsifying properties of the apo and holo forms of purified camel and bovine α-lactalbumin.

The antioxidant and antibacterial activities of camel and bovine α-lactalbumin (α-La) in both calcium-loaded (holo) and calcium-depleted (apo) forms were investigated and compared. Antioxidant assay showed that camel and bovine α-La exhibited significant Ferric-reducing antioxidant power (FRAP), ferrous iron-chelating activity (FCA) and antiradical activities especially in their apo form. Camel apo α-La also exhibited attractive antibacterial activities against Gram-negative bacteria (Pseudomonas aeruginosa) and against fungal pathogens species (Penicillium bilaiae, Aspergillus tamari and Aspergillus sclerotiorum). Likewise, emulsifying properties (emulsification ability (EAI) and stability (ESI) indexes) and the surface characteristics (surface hydrophobicity, ζ-potential and interfacial tension) of the α-La were assessed. Maximum EAI were found at pH 7.0, with higher EAI values for the camel apo α-La (EAI ~19.5 m2/g). This behavior was explained by its relative high surface hydrophobicity and its greater efficiency to reduce the surface tension at the oil-water interface. Furthermore, emulsions were found to be more stable at pH 7.0 compared to pH 5.0 (ESI ~50%) due to the higher electrostatic repulsive forces between oil droplets at pH 7.0 in consistence with the ζ-potential results. This study concluded that the camel apo α-La has antibacterial, antioxidant, and emulsifying properties in agricultural and food industries.

Isolation and Assays of Bacterial Dimethylsulfoniopropionate Lyases.

The organosulfur metabolite dimethylsulfoniopropionate (DMSP) and its enzymatic breakdown product dimethyl sulfide (DMS) have important implications in the global sulfur cycle and in marine microbial food webs. Enormous amounts of DMSP are produced in marine environments where microbial communities import and catabolize it via either the demethylation or the cleavage pathways. The enzymes that cleave DMSP are termed "DMSP lyases" and generate acrylate or hydroxypropionate, and ~107tons of DMS annually. An important environmental factor affecting DMS generation by the DMSP lyases is the availability of metal ions as these enzymes use various cofactors for catalysis. This chapter summarizes advances on bacterial DMSP catabolism, with an emphasis on various biochemical methods employed for the isolation and characterization of bacterial DMSP lyases. Strategies are presented for the purification of DMSP lyases expressed in bacterial cells. Specific conditions for the efficient isolation of apoproteins in Escherichia coli are detailed. DMSP cleavage is effectively inferred, utilizing the described HPLC-based acrylate detection assay. Finally, substrate and metal binding interactions are examined using fluorescence and UV-visible assays. Together, these methods are rapid and well suited for the biochemical and structural characterization of DMSP lyases and in the assessment of uncharacterized DMSP catabolic enzymes, and new metalloenzymes in general.

The assembly of the plant urease activation complex and the essential role of the urease accessory protein G (UreG) in delivery of nickel to urease.

Urease is a ubiquitous nickel metalloenzyme. In plants, its activation requires three urease accessory proteins (UAPs), UreD, UreF, and UreG. In bacteria, the UAPs interact with urease and facilitate activation, which involves the channeling of two nickel ions into the active site. So far this process has not been investigated in eukaryotes. Using affinity pulldowns of Strep-tagged UAPs from Arabidopsis and rice transiently expressed in planta, we demonstrate that a urease-UreD-UreF-UreG complex exists in plants and show its stepwise assembly. UreG is crucial for nickel delivery because UreG-dependent urease activation in vitro was observed only with UreG obtained from nickel-sufficient plants. This activation competence could not be generated in vitro by incubation of UreG with nickel, bicarbonate, and GTP. Compared with their bacterial orthologs, plant UreGs possess an N-terminal extension containing a His- and Asp/Glu-rich hypervariable region followed by a highly conserved sequence comprising two potential HXH metal-binding sites. Complementing the ureG-1 mutant of Arabidopsis with N-terminal deletion variants of UreG demonstrated that the hypervariable region has a minor impact on activation efficiency, whereas the conserved region up to the first HXH motif is highly beneficial and up to the second HXH motif strictly required for activation. We also show that urease reaches its full activity several days after nickel becomes available in the leaves, indicating that urease activation is limited by nickel accessibility in vivo Our data uncover the crucial role of UreG for nickel delivery during eukaryotic urease activation, inciting further investigations of the details of this process.

Kinetic regime of thermal aggregation of holo- and apoglycogen phosphorylases b.

To characterize the role of pyridoxal 5'-phosphate in stabilization of the conformation of muscle glycogen phosphorylase b (Phb), the mechanism of thermal aggregation for holo- and apoforms of Phb has been studied using dynamic light scattering. The order of aggregation with respect to the protein (n) for aggregation of holoPhb at 48°C is equal to 0.5 suggesting that the dissociative mechanism of denaturation is operative and denaturation is followed by rapid aggregation stage. In the case of aggregation of apoPhb at 37°C n=2 and the rate-limiting stage is aggregation of unfolded protein molecules.

Characterization of BciB: a ferredoxin-dependent 8-vinyl-protochlorophyllide reductase from the green sulfur bacterium Chloroherpeton thalassium.

Two enzymes, BciA and BciB, are known to reduce the C-8 vinyl group of 8-vinyl protochlorophyllide, producing protochlorophyllide a, during the synthesis of chlorophylls and bacteriochlorophylls in chlorophototrophic bacteria. BciA from the green sulfur bacterium Chlorobaculum tepidum reduces the C-8 vinyl group using NADPH as the reductant. Cyanobacteria and some other chlorophototrophs have a second, nonhomologous type of 8-vinyl reductase, BciB, but the biochemical properties of this enzyme have not yet been described. In this study, the bciB gene of the green sulfur bacterium Chloroherpeton thalassium was expressed in Escherichia coli , and the recombinant protein was purified and characterized. Recombinant BciB binds a flavin adenine dinucleotide cofactor, and EPR spectroscopy as well as quantitative analyses of bound iron and sulfide suggest that BciB binds two [4Fe-4S] clusters, one of which may not be essential for the activity of the enzyme. Using electrons provided by reduced ferredoxin or dithionite, recombinant BciB was active and reduced the 8-vinyl moiety of the substrate, 8-vinyl protochlorophyllide, producing protochlorophyllide a. A structural model for BciB based on a recent structure for the FrhB subunit of F420-reducing [NiFe]-hydrogenase of Methanothermobacter marburgensis is proposed. Possible reasons for the occurrence and distribution of BciA and BciB among various chlorophototrophs are discussed.

A manganese-rich environment supports superoxide dismutase activity in a Lyme disease pathogen, Borrelia burgdorferi.

The Lyme disease pathogen Borrelia burgdorferi represents a novel organism in which to study metalloprotein biology in that this spirochete has uniquely evolved with no requirement for iron. Not only is iron low, but we show here that B. burgdorferi has the capacity to accumulate remarkably high levels of manganese. This high manganese is necessary to activate the SodA superoxide dismutase (SOD) essential for virulence. Using a metalloproteomic approach, we demonstrate that a bulk of B. burgdorferi SodA directly associates with manganese, and a smaller pool of inactive enzyme accumulates as apoprotein. Other metalloproteins may have similarly adapted to using manganese as co-factor, including the BB0366 aminopeptidase. Whereas B. burgdorferi SodA has evolved in a manganese-rich, iron-poor environment, the opposite is true for Mn-SODs of organisms such as Escherichia coli and bakers' yeast. These Mn-SODs still capture manganese in an iron-rich cell, and we tested whether the same is true for Borrelia SodA. When expressed in the iron-rich mitochondria of Saccharomyces cerevisiae, B. burgdorferi SodA was inactive. Activity was only possible when cells accumulated extremely high levels of manganese that exceeded cellular iron. Moreover, there was no evidence for iron inactivation of the SOD. B. burgdorferi SodA shows strong overall homology with other members of the Mn-SOD family, but computer-assisted modeling revealed some unusual features of the hydrogen bonding network near the enzyme's active site. The unique properties of B. burgdorferi SodA may represent adaptation to expression in the manganese-rich and iron-poor environment of the spirochete.

Effect of pH on the catalytic function and zinc content of native and immobilized anthrax lethal factor.

Translocation of the zinc-dependent metalloendopeptidase anthrax lethal factor (LF) from the endosome to the cytosol requires an acidic endosomal milieu. In the current study, we utilized immobilized (to prevent protein aggregation below pH 5.5) and native LF to assess the effect of pH on the function and metal content of LF. Our results reveal the diminution of LF's catalytic competence under moderately acidic conditions (pH ∼6) to be uncorrelated to the metal content of the protein. However, a significant degree of demetallation of LF (∼30%) was observed at pH values close to those found in late endosomes (pH ∼5), thus raising the possibility that a substantial proportion of LF molecules may not be in their zinc-bound state prior to translocation.

Crystallization and preliminary crystallographic analysis of 2-aminophenol 1,6-dioxygenase complexed with substrate and with an inhibitor.

Dioxygen activation implemented by nonhaem Fe(II) enzymes containing the 2-His-1-carboxylate facial triad has been extensively studied in recent years. Extradiol dioxygenase is the archetypal member of this superfamily and catalyzes the oxygenolytic ring opening of catechol analogues. Here, the crystallization and preliminary X-ray analysis of 2-aminophenol 1,6-dioxygenase, an enzyme representing a minor subset of extradiol dioxygenases that catalyze the fission of 2-aminophenol rather than catecholic compounds, is reported. Crystals of the holoenzyme with FeII and of complexes with the substrate 2-aminophenol and the suicide inhibitor 4-nitrocatechol were grown using the cocrystallization method under the same conditions as used for the crystallization of the apoenzyme. The crystals belonged to space group C2 and diffracted to 2.3-2.7 Šresolution; the crystal that diffracted to the highest resolution had unit-cell parameters a=270.24, b=48.39, c=108.55 Å, ß=109.57°. All X-ray data sets collected from diffraction-quality crystals were suitable for structure determination.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the apo form of InsP5 2-K from Arabidopsis thaliana.

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP(5) 2-K) is a key enzyme that catalyzes the synthesis of phytic acid (IP(6)) from inositol 1,3,4,5,6-pentakisphosphate (IP(5)) and ATP. The first structure of IP(5) 2-K, that from Arabidopsis thaliana, has been solved previously; it only crystallized in the presence of inositol, either the substrate IP(5) or the product IP(6), and failed to crystallize in its free state (without inositol). Based on structural analysis, a point mutation of IP(5) 2-K (W129A) has been produced in order to overcome this limitation and obtain information about protein conformational changes upon substrate binding. Here, the production and crystallization of W129A IP(5) 2-K in its free state and with bound nucleotide is described. These crystals differed from the native crystals and belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 66.00, b = 68.23, c = 105.80 Å and a = 63.06, b = 71.80, c = 100.23 Å, respectively. The crystals diffracted to resolutions of 2.22 Å (apo) and 2.05 Å (nucleotide bound) using synchrotron radiation and contained one molecule per asymmetric unit. The structures have been determined using the molecular-replacement method and refinement is being undertaken.

Amyloid fibrillation in native and chemically-modified forms of carbonic anhydrase II: role of surface hydrophobicity.

Chemical modification or mutation of proteins may bring about significant changes in the net charge or surface hydrophobicity of a protein structure. Such events may be of major physiological significance and may provide important insights into the genetics of amyloid diseases. In the present study, fibrillation potential of native and chemically-modified forms of bovine carbonic anhydrase II (BCA II) were investigated. Initially, various denaturing conditions including low pH and high temperatures were tested to induce fibrillation. At a low pH of around 2.4, where the protein is totally dissociated, the apo form was found to take up a pre-molten globular (PMG) conformation with the capacity for fibril formation. Upon increasing the pH to around 3.6, a molten globular (MG) form became abundant, forming amorphous aggregates. Charge neutralization and enhancement of hydrophobicity by methylation, acetylation and propionylation of lysine residues appeared very effective in promoting fibrillation of both the apo and holo forms under native conditions, the rates and extents of which were directly proportional to surface hydrophobicity, and influenced by salt concentration and temperature. These modified structures underwent more pronounced fibrillation under native conditions, than the PMG intermediate form, observed under denaturing conditions. The nature of the fibrillation products obtained from intermediate and modified structures were characterized and compared and their possible cytotoxicity determined. Results are discussed in terms of the importance of surface net charge and hydrophobicity in controlling protein aggregation. A discussion on the physiological significance of the observations is also presented.

Structural and biochemical studies of human 4-hydroxy-2-oxoglutarate aldolase: implications for hydroxyproline metabolism in primary hyperoxaluria.

BACKGROUND: 4-hydroxy-2-oxoglutarate (HOG) aldolase is a unique enzyme in the hydroxyproline degradation pathway catalyzing the cleavage of HOG to pyruvate and glyoxylate. Mutations in this enzyme are believed to be associated with the excessive production of oxalate in primary hyperoxaluria type 3 (PH3), although no experimental data is available to support this hypothesis. Moreover, the identity, oligomeric state, enzymatic activity, and crystal structure of human HOGA have not been experimentally determined. METHODOLOGY/PRINCIPAL FINDINGS: In this study human HOGA (hHOGA) was identified by mass spectrometry of the mitochondrial enzyme purified from bovine kidney. hHOGA performs a retro-aldol cleavage reaction reminiscent of the trimeric 2-keto-3-deoxy-6-phosphogluconate aldolases. Sequence comparisons, however, show that HOGA is related to the tetrameric, bacterial dihydrodipicolinate synthases, but the reaction direction is reversed. The 1.97 Å resolution crystal structure of hHOGA bound to pyruvate was determined and enabled the modeling of the HOG-Schiff base intermediate and the identification of active site residues. Kinetic analyses of site-directed mutants support the importance of Lys196 as the nucleophile, Tyr168 and Ser77 as components of a proton relay, and Asn78 and Ser198 as unique residues that facilitate substrate binding. CONCLUSIONS/SIGNIFICANCE: The biochemical and structural data presented support that hHOGA utilizes a type I aldolase reaction mechanism, but employs novel residue interactions for substrate binding. A mapping of the PH3 mutations identifies potential rearrangements in either the active site or the tetrameric assembly that would likely cause a loss in activity. Altogether, these data establish a foundation to assess mutant forms of hHOGA and how their activity could be pharmacologically restored.

Expression, purification and characterization of arginase from Helicobacter pylori in its apo form.

Arginase, a manganese-dependent enzyme that widely distributed in almost all creatures, is a urea cycle enzyme that catalyzes the hydrolysis of L-arginine to generate L-ornithine and urea. Compared with the well-studied arginases from animals and yeast, only a few eubacterial arginases have been characterized, such as those from H. pylori and B. anthracis. However, these enzymes used for arginase activity assay were all expressed with LB medium, as low concentration of Mn(2+) was detectable in the medium, protein obtained were partially Mn(2+) bonded, which may affect the results of arginase activity assay. In the present study, H. pylori arginase (RocF) was expressed in a Mn(2+) and Co(2+) free minimal medium, the resulting protein was purified through affinity and gel filtration chromatography and the apo-form of RocF was confirmed by flame photometry analysis. Gel filtration indicates that the enzyme exists as monomer in solution, which was unique as compared with homologous enzymes. Arginase activity assay revealed that apo-RocF had an acidic pH optimum of 6.4 and exhibited metal preference of Co(2+)>Ni(2+)>Mn(2+). We also confirmed that heat-activation and reducing regents have significant impact on arginase activity of RocF, and inhibits S-(2-boronoethyl)-L-Cysteine (BEC) and Nω-hydroxy-nor-Arginine (nor-NOHA) inhibit the activity of RocF in a dose-dependent manner.

Crystal structures of Burkholderia cenocepacia dihydropteroate synthase in the apo-form and complexed with the product 7,8-dihydropteroate.

BACKGROUND: The enzyme dihydropteroate synthase (DHPS) participates in the de novo synthesis of folate cofactors by catalyzing the formation of 7,8-dihydropteroate from condensation of p-aminobenzoic acid with 6-hydroxymethyl-7,8-dihydropteroate pyrophosphate. DHPS is absent from humans, who acquire folates from diet, and has been validated as an antimicrobial therapeutic target by chemical and genetic means. The bacterium Burkholderia cenocepacia is an opportunistic pathogen and an infective agent of cystic fibrosis patients. The organism is highly resistant to antibiotics and there is a recognized need for the identification of new drugs against Burkholderia and related Gram-negative pathogens. Our characterization of the DHPS active site and interactions with the enzyme product are designed to underpin early stage drug discovery. RESULTS: An efficient recombinant protein expression system for DHPS from B. cenocepacia (BcDHPS) was prepared, the dimeric enzyme purified in high yield and crystallized. The structure of the apo-enzyme and the complex with the product 7,8-dihydropteroate have been determined to 2.35 Å and 1.95 Å resolution respectively in distinct orthorhombic crystal forms. The latter represents the first crystal structure of the DHPS-pterin product complex, reveals key interactions involved in ligand binding, and reinforces data generated by other structural studies. Comparisons with orthologues identify plasticity near the substrate-binding pocket and in particular a range of loop conformations that contribute to the architecture of the DHPS active site. These structural data provide a foundation for hit discovery. An intriguing observation, an artifact of the analysis, that of a potential sulfenamide bond within the ligand complex structure is mentioned. CONCLUSION: Structural similarities between BcDHPS and orthologues from other Gram-negative species are evident as expected on the basis of a high level of sequence identity. The presence of 7,8-dihydropteroate in the binding site provides details about ligand recognition by the enzyme and the different states of the enzyme allow us to visualize distinct conformational states of loops adjacent to the active site. Improved drugs to combat infections by Burkholderia sp. and related Gram-negative bacteria are sought and our study now provides templates to assist that process and allow us to discuss new ways of inhibiting DHPS.

Crystallization and preliminary X-ray crystallographic studies of enoyl-acyl carrier protein reductase (FabI) from Psuedomonas aeruginosa.

During fatty-acid biosynthesis, enoyl-acyl carrier protein (enoyl-ACP) reductase catalyzes the reduction of trans-2-enoyl-ACP to fully saturated acyl-ACP via the ubiquitous fatty-acid synthase system. NADH-dependent enoyl-ACP reductase (FabI) from Pseudomonas aeruginosa has been purified and crystallized as an apoenzyme and in a complex form with NADH and triclosan. Triclosan is an inhibitor of FabI and forms a stable ternary complex in the presence of NADH. The crystals of native and complexed FabI diffracted to resolutions of 2.6 and 1.8 Å, respectively. The crystals both belonged to space group P2(1), with unit-cell parameters a = 117.32, b = 155.844, c = 129.448 Å, ß = 111.061° for the native enzyme and a = 64.784, b = 107.573, c = 73.517 Å, ß = 116.162° for the complex. Preliminary molecular replacement further confirmed the presence of four tetramers of native FabI and one tetramer of the complex in the asymmetric unit, corresponding to Matthews coefficients (V(M)) of 2.46 and 2.05 Å(3) Da(-1) and solvent contents of 50.1 and 40.1%, respectively.

Crystal structures of apo and metal-bound forms of the UreE protein from Helicobacter pylori: role of multiple metal binding sites.

The crystal structure of the urease maturation protein UreE from Helicobacter pylori has been determined in its apo form at 2.1 A resolution, bound to Cu(2+) at 2.7 A resolution, and bound to Ni(2+) at 3.1 A resolution. Apo UreE forms dimers, while the metal-bound enzymes are arranged as tetramers that consist of a dimer of dimers associated around the metal ion through coordination by His102 residues from each subunit of the tetramer. Comparison of independent subunits from different crystal forms indicates changes in the relative arrangement of the N- and C-terminal domains in response to metal binding. The improved ability of engineered versions of UreE containing hexahistidine sequences at either the N-terminal or C-terminal end to provide Ni(2+) for the final metal sink (urease) is eliminated in the H102A version. Therefore, the ability of the improved Ni(2+)-binding versions to deliver more nickel is likely an effect of an increased local concentration of metal ions that can rapidly replenish transferred ions bound to His102.

Purification, crystallization and preliminary X-ray analysis of apo glyceraldehyde-3-phosphate dehydrogenase 1 (GAP1) from methicillin-resistant Staphylococcus aureus (MRSA252).

Glyceraldehyde-3-phosphate dehydrogenase 1 (GAP1) from methicillin-resistant Staphylococcus aureus (MRSA252) has been purified to homogeneity in the apo form. The protein was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group P2(1), with unit-cell parameters a = 69.95, b = 93.68, c = 89.05 A, beta = 106.84 degrees . X-ray diffraction data have been collected and processed to a maximum resolution of 2.2 A. The presence of one tetramer in the asymmetric unit gives a Matthews coefficient (V(M)) of 1.81 A(3) Da(-1) with a solvent content of 32%. The structure has been solved by molecular replacement and structure refinement is now in progress.

Self-subunit swapping chaperone needed for the maturation of multimeric metalloenzyme nitrile hydratase by a subunit exchange mechanism also carries out the oxidation of the metal ligand cysteine residues and insertion of cobalt.

The incorporation of cobalt into low molecular mass nitrile hydratase (L-NHase) of Rhodococcus rhodochrous J1 has been found to depend on the alpha-subunit exchange between cobalt-free L-NHase (apo-L-NHase lacking oxidized cysteine residues) and its cobalt-containing mediator (holo-NhlAE containing Cys-SO(2)(-) and Cys-SO(-) metal ligands), this novel mode of post-translational maturation having been named self-subunit swapping, and NhlE having been recognized as a self-subunit swapping chaperone (Zhou, Z., Hashimoto, Y., Shiraki, K., and Kobayashi, M. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 14849-14854). We discovered here that cobalt was inserted into both the cobalt-free NhlAE (apo-NhlAE) and the cobalt-free alpha-subunit (apo-alpha-subunit) in an NhlE-dependent manner in the presence of cobalt and dithiothreitol in vitro. Matrix-assisted laser desorption ionization time-of-flight mass spectroscopy analysis revealed that the non-oxidized cysteine residues in apo-NhlAE were post-translationally oxidized after cobalt insertion. These findings suggested that NhlE has two activities, i.e. cobalt insertion and cysteine oxidation. NhlE not only functions as a self-subunit swapping chaperone but also a metallochaperone that includes a redox function. Cobalt insertion and cysteine oxidation occurred under both aerobic and anaerobic conditions when Co(3+) was used as a cobalt donor, suggesting that the oxygen atoms in the oxidized cysteines were derived from water molecules but not from dissolved oxygen. Additionally, we isolated apo-NhlAE after the self-subunit swapping event and found that it was recycled for cobalt transfer into L-NHase.

Understanding the structural basis for substrate and inhibitor recognition in eukaryotic GH11 xylanases.

Endo-beta1,4-xylanases (xylanases) hydrolyse the beta1,4 glycosidic bonds in the backbone of xylan. Although xylanases from glycoside hydrolase family 11 (GH11) have been extensively studied, several issues remain unresolved. Thus, the mechanism by which these enzymes hydrolyse decorated xylans is unclear and the structural basis for the variation in catalytic activity within this family is unknown. Furthermore, the mechanism for the differences in the inhibition of fungal GH11 enzymes by the wheat protein XIP-I remains opaque. To address these issues we report the crystal structure and biochemical properties of the Neocallimastix patriciarum xylanase NpXyn11A, which displays unusually high catalytic activity and is one of the few fungal GH11 proteins not inhibited by XIP-I. Although the structure of NpXyn11A could not be determined in complex with substrates, we have been able to investigate how GH11 enzymes hydrolyse decorated substrates by solving the crystal structure of a second GH11 xylanase, EnXyn11A (encoded by an environmental DNA sample), bound to ferulic acid-1,5-arabinofuranose-alpha1,3-xylotriose (FAX(3)). The crystal structure of the EnXyn11A-FAX(3) complex shows that solvent exposure of the backbone xylose O2 and O3 groups at subsites -3 and +2 allow accommodation of alpha1,2-linked 4-methyl-D-glucuronic acid and L-arabinofuranose side chains. Furthermore, the ferulated arabinofuranose side chain makes hydrogen bonds and hydrophobic interactions at the +2 subsite, indicating that the decoration may represent a specificity determinant at this aglycone subsite. The structure of NpXyn11A reveals potential -3 and +3 subsites that are kinetically significant. The extended substrate-binding cleft of NpXyn11A, compared to other GH11 xylanases, may explain why the Neocallimastix enzyme displays unusually high catalytic activity. Finally, the crystal structure of NpXyn11A shows that the resistance of the enzyme to XIP-I is not due solely to insertions in the loop connecting beta strands 11 and 12, as suggested previously, but is highly complex.

Metalation of the amyotrophic lateral sclerosis mutant glycine 37 to arginine superoxide dismutase (SOD1) apoprotein restores its structural and dynamical properties in solution to those of metalated wild-type SOD1.

The G37R copper-zinc superoxide dismutase (SOD1) is one of the many mutant SOD1 proteins known to cause familial amyotrophic lateral sclerosis by an unknown mechanism. This particular mutation occurs in the beta barrel plug, a region proposed to be critical for the structural stability of the protein. The behavior of G37R asSOD1 was studied in solution where it was observed that, when the protein is fully metalated, its global structure, mobility, and stability are virtually indistinguishable from those of the nonmutated protein. By contrast, although the presence of the G37R mutation does not result in a substantial change of the overall structure of the metal-free apoprotein in solution, it does affect the key conformational features of the beta-barrel plug such that (i) apo G37R asSOD1 melts at a temperature approximately 10 degrees C lower than apo asSOD1, (ii) it aggregates more rapidly than apo asSOD1, and (iii) interaction with trifluoroethanol (TFE) can deform it into a structure with a much higher degree of alpha-helical content. The increased plasticity of the apo G37R asSOD1 mutant protein is likely responsible for its enhanced tendency to aggregate in concentrated solutions. These results suggest further that it is the metal-free apo forms of the mutant SOD1 protein that are the agents of its toxicity.

Cox17, a copper chaperone for cytochrome c oxidase: expression, purification, and formation of mixed disulphide adducts with thiol reagents.

Copper chaperone for cytochrome c oxidase (Cox17) is a 7 kDa copper-binding protein, which facilitates incorporation of copper ions into Cu(A) site of cytochrome c oxidase. Cox17 contains six conserved Cys residues and occurs in three different oxidative states, which display different metal-binding properties and stability. In the present study, we have elaborated technologies for production of partially oxidized human recombinant Cox17 in a bacterial expression system and purification of fully oxidized Cox17. For this purpose we used Escherichia coli Origami strain, which is deficient in thioredoxin and thioredoxin reductase systems and allows formation of disulfide bonds in cytoplasmic proteins. Fully oxidized Cox17 was purified by a simplified two-step procedure including gel filtration and cation exchange chromatography. By using mass spectrometry we demonstrated that application of 2-mercaptoethanol (2-ME) during purification leads to formation of its mixed disulfide adducts with Cox17. Moreover, partially reduced Cox17 can form mixed disulfide adducts also with the cellular reducing agent glutathione, which abolishes copper-binding ability of partially reduced Cox17.