Nickel binding and immunological properties of the C-terminal domain of the Helicobacter pylori GroES homologue (HspA)

Helicobacter pylori synthesizes a heat-shock protein of the GroES class. The gene encoding this protein (heat-shock protein A, HspA) was recently cloned and it was shown to be unique in structure. H. pylori HspA consists of two domains: the N-terminal domain (domain A) homologous with other GroES proteins, and a C-terminal domain (domain B) corresponding to 27 additional residues resembling a metal-binding domain. Various recombinant proteins consisting of the entire HspA polypeptide, the A domain, or the B domain were produced independently as proteins fused to maltose-binding protein (MBP). Comparison of the divalent cation binding properties of the various MBP and MBP-fused proteins allowed us to conclude that HspA binds nickel ions by means of its C-terminal domain. HspA exhibited a high and specific affinity for nickel ions in comparison with its affinity for other divalent cations (copper, zinc, cobalt). Equilibrium dialysis experiments revealed that MBP–HspA binds nickel ions with an apparent dissociation constant (K_d) of 1.8 μM and a stoichiometry of 1.9 ions per molecule. The analysis of the deduced HspA amino acid sequences encoded by 35 independent clinical isolates demonstrated the existence of two molecular variants of HspA, i.e. a major and a minor variant present in 89% and 11% of strains, respectively. The two variants differed from each other by the simultaneous substitution of seven amino acids within the B domain, whilst the A domain was highly conserved amongst all the HspA proteins (99–100% identity). On the basis of serological studies, the highly conserved A domain of HspA was found to be the immunodominant domain. Functional complementation experiments were performed to test the properties of the two HspA variants. When co-expressed together with the H. pylori urease gene cluster in Escherichia coli cells, the two HspA variant-encoding genes led to a fourfold increase in urease activity, demonstrating that HspA in H. pylori has a specialized function with regard to the nickel metalloenzyme urease.     

Metallo-GTPase HypB from Helicobacter pylori and its interaction with nickel chaperone protein HypA

The maturation of [NiFe]-hydrogenase is highly dependent on a battery of chaperone proteins. Among these, HypA and HypB were proposed to exert nickel delivery functions in the metallocenter assembly process, although the detailed mechanism remains unclear. Herein, we have overexpressed and purified wild-type HypB as well as two mutants, K168A and M186L/F190V, from Helicobacter pylori. We demonstrated that all proteins bind Ni(2+) at a stoichiometry of one Ni(2+) per monomer of the proteins with dissociation constants at micromolar levels. Ni(2+) elevated GTPase activity of WT HypB, which is attributable to a lower affinity of the protein toward GDP as well as Ni(2+)-induced dimerization. The disruption of GTP-dependent dimerization has led to GTPase activities of both mutants in apo-forms almost completely abolished, compared with the wild-type protein. The GTPase activity is partially restored for HypB(M186L/F190V) mutant but not for HypB(K168A) mutant upon Ni(2+) binding. HypB forms a complex with its partner protein HypA with a low affinity (K(d) of 52.2 ± 8.8 μM). Such interactions were also observed in vivo both in the absence and presence of nickel using a GFP-fragment reassembly technique. The putative protein-protein interfaces on H. pylori HypA and HypB proteins were identified by NMR chemical shift perturbation and mutagenesis studies, respectively. Intriguingly, the unique N terminus of H. pylori HypB was identified to participate in the interaction with H. pylori HypA. These structural and functional studies provide insight into the molecular mechanism of Ni(2+) delivery during maturation of [NiFe]-hydrogenase.     

Interactions of bismuth complexes with metallothionein(II).

Bismuth complexes are widely used as anti-ulcer drugs and can significantly reduce the side effects of platinum anti-cancer drugs. Bismuth is known to induce the synthesis of metallothionein (MT) in the kidney, but there are few chemical studies on the interactions of bismuth complexes with metallothionein. Here we show that Bi(3+) binds strongly to metallothionein with a stoichiometry bismuth:MT = 7:1 (Bi(7)MT) and can readily displace Zn(2+) and Cd(2+). Bismuth is still bound to the protein even in strongly acidic solutions (pH 1). Reactions of bismuth citrate with MT are faster than those of [Bi(EDTA)](-), and both exhibit biphasic kinetics. (1)H NMR data show that Zn(2+) is displaced faster than Cd(2+), and that both Zn(2+) and Cd(2+) in the beta-domain (three metal cluster) of MT are displaced by Bi(3+) much faster than from the alpha-domain (four metal cluster). The extended x-ray absorption fine structure spectrum of Bi(7)MT is very similar to that for the glutathione and N-acetyl-L-cysteine complexes [Bi(GS)(3)] and [Bi(NAC)(3)] with an inner coordination sphere of three sulfur atoms and average Bi-S distances of 2.55 A. Some sites appear to contain additional short Bi-O bonds of 2.2 A and longer Bi-S bonds of 3.1 A. The Bi(3+) sites in Bi(7)MT are therefore highly distorted in comparison with those of Zn(2+) and Cd(2+).     

幽门螺杆菌热休克蛋白A基因的克隆、表达及免疫原性研究

幽门螺杆菌的感染可诱发人体产生胃炎和消化性溃疡,其组成成分热休克蛋白Ａ（ＨｓｐＡ）可刺激机体产生保护性的免疫反应。用ＰＣＲ方法从幽门螺杆菌的染色体ＤＮＡ上扩增出ＨｓｐＡ基因片段,将其插入原核表达载体ｐＥＴ２２ｂ（＋）中,并在ＢＬ２１（ＤＥ３）大肠杆菌表达。经测序ＨｓｐＡ基因片段有３５４ｂｐ组成,可编码１１８个氨基酸残基的多肽。ＳＤＳＰＡＧＥ和免疫印迹分析检测发现,ＨｓｐＡ基因表达的蛋白质分子量约为１５ｋＤ,并证实该重组蛋白质可以被幽门螺杆菌感染阳性患者的血清所识别,同时将其免疫小鼠可刺激机体产生抗该重组蛋白质的抗体。ＨｓｐＡ有可能作为一种有效的蛋白质疫苗用于幽门螺杆菌感染的预防和治疗。     

幽门螺杆菌HspA融合蛋白口服疫苗的构建

构建表达幽门螺杆菌的保护性抗原分热休克蛋白A亚单位(HspA）和霍乱毒素B亚单位（CtxB）的重组融合蛋白的生物工程菌株,以此制备幽门螺杆菌的口服疫苗。用PCR方法扩增hspA和ctxB两个目的的基因片段,将它们分别克隆至pSK（＋）质粒上,然后插入含T7启动子ET－22b（＋）的表达载体中,构建嗓基因的表达质量pET－hct,转化E.coliBL21(DE3）,经IPTG诱导表达融合蛋白HCT。经测序,hspA-ctxB(hct)融合基因片段由726bp组成,可以编码242个氨基酸残基的多肽。经SDS－PAGE和免疫印迹分析检测发现,融合基因表达的蛋白质相对分子质量约为30kD。融合蛋白经镍离子柱纯化、复性后,和HspA共同标记同位素^125I,然后给小鼠灌胃,结果观察到HCT组小鼠血清中的^125I的放射量要明显高于HspA组（P＜0．001）,且吸收峰值时间明显提前。融合蛋白中的CtxB可明显促进小鼠对HspA的吸收,HCT融合蛋白可以作为预防和治疗幽门螺杆菌感染的侯选口服疫苗。     

Zn2+-linked dimerization of UreG from Helicobacter pylori, a chaperone involved in nickel trafficking and urease activation

The biosynthesis of the active metal-bound form of the nickel-dependent enzyme urease involves the formation of a lysine-carbamate functional group concomitantly with the delivery of two Ni(2+) ions into the precast active site of the apoenzyme and with GTP hydrolysis. In the urease system, this role is performed by UreG, an accessory protein belonging to the group of homologous P-loop GTPases, often required to complete the biosynthesis of nickel-enzymes. This study is focused on UreG from Helicobacter pylori (HpUreG), a bacterium responsible for gastric ulcers and cancer, infecting large part of the human population, and for which urease is a fundamental virulence factor. The soluble HpUreG was expressed in E. coli and purified to homogeneity. On-line size exclusion chromatography and light scattering indicated that apo-HpUreG exists as a monomer in solution. Circular dichroism, which demonstrated the presence of a well-defined secondary structure, and NMR spectroscopy, which revealed a large number of residues that appear structured on the basis of their backbone amide proton chemical shift dispersion, indicated that, at variance with other UreG proteins so far characterized, this protein is significantly folded in solution. The amino acid sequence of HpUreG is 29% identical to that of HypB from Methanocaldococcus jannaschii, a dimeric zinc-binding GTPase involved in the in vivo assembly of [Ni,Fe]-hydrogenase. A homology-based molecular model of HpUreG was calculated, which allowed us to identify structural and functional features of the protein. Isothermal titration microcalorimetry demonstrated that HpUreG specifically binds 0.5 equivalents of Zn(2+) per monomer (K(d) = 0.33 +/- 0.03 microM), whereas it has 20-fold lower affinity for Ni(2+) (K(d) = 10 +/- 1 microM). Zinc ion binding (but not Ni(2+) binding) causes protein dimerization, as confirmed using light scattering measurements. The structural rearrangement occurring upon Zn(2+)-binding and consequent dimerization was evaluated using circular dichroism and fluorescence spectroscopy. Fully conserved histidine and cysteine residues were identified and their role in zinc binding was verified by site-directed mutagenesis and microcalorimetry. The results are analyzed and discussed with respect to analogous examples of GTPases in nickel metabolism.     

In vivo and in vitro kinetics of metal transfer by the Klebsiella aerogenes urease nickel metallochaperone, UreE

The urease accessory protein encoded by ureE from Klebsiella aerogenes is proposed to deliver Ni(II) to the urease apoprotein during enzyme activation. Native UreE possesses a histidine-rich region at its carboxyl terminus that binds several equivalents of Ni(2+); however, a truncated form of this protein (H144*UreE) binds only 2 Ni(2+) per dimer and is functionally active (Brayman, T. G., and Hausinger, R. P. (1996) J. Bacteriol. 178, 5410-5416). The urease activation kinetics were studied in vivo by monitoring the development of urease activity upon adding Ni(2+) to spectinomycin-treated Escherichia coli cells that expressed the complete K. aerogenes urease gene cluster with altered forms of ureE. Site-specific alterations of H144*UreE decrease the rate of in vivo urease activation, with the most dramatic changes observed for the H96A, H110A, D111A, and H112A substitutions. Notably, urease activity in cells producing H96A/H144*UreE was lower than cells containing a ureE deletion. Prior studies had shown that H110A and H112A variants each bound a single Ni(2+) per dimer with elevated K(d) values compared with control H144*UreE, whereas the H96A and D111A variants bound 2 Ni(2+) per dimer with unperturbed K(d) values (Colpas, G. J., Brayman, T. G., Ming, L.-J., and Hausinger, R. P. (1999) Biochemistry 38, 4078-4088). To understand why cells containing the latter two proteins showed reduced rates of urease activation, we characterized their metal binding/dissociation kinetics and compared the results to those obtained for H144*UreE. The truncated protein was shown to sequentially bind two Ni(2+) with k(1) approximately 18 and k(2) approximately 100 M(-1) s(-1), and with dissociation rates k(-1) approximately 3 x 10(-3) and k(-2) approximately 10(-4) s(-1). Similar apparent rates of binding and dissociation were noted for the two mutant proteins, suggesting that altered H144*UreE interactions with Ni(2+) do not account for the changes in cellular urease activation. These conclusions are further supported by in vitro experiments demonstrating that addition of H144*UreE to urease apoprotein activation mixtures inhibited the rate and extent of urease formation. Our results highlight the importance of other urease accessory proteins in assisting UreE-dependent urease maturation.     

Competitive binding of bismuth to transferrin and albumin in aqueous solution and in blood plasma

Several bismuth compounds are currently used as antiulcer drugs, but their mechanism of action is not well established. Proteins are thought to be target sites. In this work we establish that the competitive binding of Bi(3+) to the blood serum proteins albumin and transferrin, as isolated proteins and in blood plasma, can be monitored via observation of (1)H and (13)C NMR resonances of isotopically labeled [epsilon-(13)C]Met transferrin. We show that Met(132) in the I132M recombinant N-lobe transferrin mutant is a sensitive indicator of N-lobe metal binding. Bi(3+) binds to the specific Fe(3+) sites of transferrin and the observed shifts of Met resonances suggest that Bi(3+) induces similar conformational changes in the N-lobe of transferrin in aqueous solution and plasma. Bi(3+) binding to albumin is nonspecific and Cys(34) is not a major binding site, which is surprising because Bi(3+) has a high affinity for thiolate sulfur. This illustrates that the potential target sites for metals (in this case Bi(3+)) in proteins depend not only on their presence but also on their accessibility. Bi(3+) binds to transferrin in preference to albumin both in aqueous solution and in blood plasma.     

幽门螺杆菌热休克蛋白A在乳酸乳球菌中的表达及免疫学活性分析

目的构建含幽门螺杆菌(H.pylori)热休克蛋白A编码基因的重组载体,并电转入乳酸乳球菌MG1363,表达目的蛋白并分析其免疫原性,为H.pylori基因工程口服疫苗的研究和开发奠定基础。方法以H.py-loriNCTC 11637株基因组DNA为模板,PCR扩增hspA基因,并克隆至乳酸乳球菌表达载体pMG36e中。将重组质粒转化E.coliDH5α,经鉴定的阳性重组质粒命名为pMG36e/hspA。以电穿孔法将pMG36e/hspA转化乳酸乳球菌MG1363并用Western blot检测HspA蛋白的表达。结果克隆重组后得到pMG36e/hspA。将pMG36e/hspA电转化MG1363后,收集菌体蛋白进行Western blot分析,在HspA的相对分子质量(Mr≈13 kDa)处出现特异性条带。结论首次成功构建了表达H.pyloriHspA的重组乳酸乳球菌MG1363,为进一步口服疫苗的相关研究奠定了基础。     

Inlaid Nd-substituted bismuth titanate nanoplates for protein immobilization and Nd-controlled electrochemical properties

Neodymium (Nd) substituted bismuth titanate (Bi(4-x)Nd(x)Ti(3)O(12), BNTO-x) nanoplates inlaid one another were prepared by sol-gel hydrothermal method, which was explored for protein immobilization and biosensor fabrication. Comparative experiments witnessed that Bi(3+) ions in bismuth titanate (Bi(4)Ti(3)O(12), BTO) were successfully substituted with Nd(3+) ions, and the electrochemical properties of the Hb-Chi-BNTO biosensors closely depended on the Nd(3+) ion content. With increasing the Nd(3+) doping content, the electrochemical performance of the Hb-Chi-BNTO-x biosensors showed regularly variable. Moreover, compared with the Hb-Chi-BTO and other Hb-Chi-BNTO-x biosensors, the Hb-Chi-BNTO-0.85 biosensor had more excellent electrochemical and electrocatalytic properties such as stronger redox peak currents (approximately three-fold), smaller peak-to-peak separation (50 mV), larger heterogeneous electron transfer rate (14.1 ± 3.8s(-1)), higher surface concentration of electroactive redox protein (about 8.16 × 10(-11)mol/cm(2)), and better reproducibility and stability. The Nd-depended electrochemical properties of the Hb-Chi-BNTO biosensors may open up a new idea for designing third-generation electrochemical biosensors, and the BNTO-0.85-based biosensor is also expected to find potential applications in many areas such as biomedical, food, and environmental detection.     

Helicobacter pylori ABC transporter: effect of allelic exchange mutagenesis on urease activity.

Helicobacter pylori urease requires nickel ions in the enzyme active site for catalytic activity. Nickel ions must, therefore, be actively acquired by the bacterium. NixA (high-affinity nickel transport protein)-deficient mutants of H. pylori retain significant urease activity, suggesting the presence of alternate nickel transporters. Analysis of the nucleotide sequence of the H. pylori genome revealed a homolog of NikD, a component of an ATP-dependent nickel transport system in Escherichia coli. Based on this sequence, a 378-bp DNA fragment was PCR amplified from H. pylori genomic DNA and used as a probe to identify an H. pylori lambda ZAPII genomic library clone that carried these sequences. Four open reading frames of 621, 273, 984, and 642 bp (abcABCD) were revealed by sequencing and predicted polypeptides of 22.7, 9.9, 36.6, and 22.8 kDa, respectively. The 36.6-kDa polypeptide (AbcC) has significant homology (56% amino acid sequence identity) to an E. coli ATP-binding protein component of an ABC transport system, while none of the other putative proteins are significantly homologous to polypeptides in the available databases. To determine the possible contribution of these genes to urease activity, abcC and abcD were each insertionally inactivated with a kanamycin resistance (aphA) cassette and allelic exchange mutants of each gene were constructed in H. pylori UMAB41. Mutation of abcD resulted in an 88% decrease in urease activity to 27 +/- 31 mumol of NH3/min/mg of protein (P < 0.0001), and a double mutant of nixA and abcC resulted in the near abolishment of urease activity (1.1 +/- 1.4 mumol of NH3/min/mg of protein in the double mutant versus 228 +/- 92 mumol of NH3/min/mg of protein in the parent [P < 0.0001]). Synthesis of urease apoenzyme, however, was unaffected by mutations in any of the abc genes. We conclude that the abc gene cluster, in addition to nixA, is involved in production of a catalytically active urease.     

Biochemical and structural studies on native and recombinant Glycine max UreG: a detailed characterization of a plant urease accessory protein

Urea is the nitrogen fertilizer most utilized in crop production worldwide. Understanding all factors involved in urea metabolism in plants is an essential step towards assessing and possibly improving the use of urea by plants. Urease, the enzyme responsible for urea hydrolysis, and its accessory proteins, necessary for nickel incorporation into the enzyme active site and concomitant activation, have been extensively characterized in bacteria. In contrast, little is known about their plant counterparts. This work reports a detailed characterization of Glycine max UreG (GmUreG), a urease accessory protein. Two forms of native GmUreG, purified from seeds, were separated by metal affinity chromatography, and their properties (GTPase activity in absence and presence of Ni(2+) or Zn(2+), secondary structure and metal content) were compared with the recombinant protein produced in Escherichia coli. The binding affinity of recombinant GmUreG (rGmUreG) for Ni(2+) and Zn(2+) was determined by isothermal titration calorimetry. rGmUreG binds Zn(2+) or Ni(2+) differently, presenting a very tight binding site for Zn(2+) (K (d) = 0.02 ± 0.01 μM) but not for Ni(2+), thus suggesting that Zn(2+) may play a role on the plant urease assembly process, as suggested for bacteria. Size exclusion chromatography showed that Zn(2+) stabilizes a dimeric form of the rGmUreG, while NMR measurements indicate that rGmUreG belongs to the class of intrinsically disordered proteins. A homology model for the fully folded GmUreG was built and compared to bacterial UreG models, and the possible sites of interaction with other accessory proteins were investigated.     

In HspA from Helicobacter pylori vicinal disulfide bridges are a key determinant of domain B structure

Helicobacter pylori produces a heat shock protein A (HspA) that is unique to this bacteria. While the first 91 residues (domain A) of the protein are similar to GroES, the last 26 (domain B) are unique to HspA. Domain B contains eight histidines and four cysteines and was suggested to bind nickel. We have produced HspA and two mutants: Cys94Ala and Cys94Ala/Cys111Ala and identified the disulfide bridge pattern of the protein. We found that the cysteines are engaged in three disulfide bonds: Cys51/Cys53, Cys94/Cys111 and Cys95/Cys112 that result in a unique closed loop structure for the domain B.     

Escherichia coli HypA is a zinc metalloprotein with a weak affinity for nickel

The hyp operon encodes accessory proteins that are required for the maturation of the [NiFe] hydrogenase enzymes and, in some organisms, for the production of urease enzymes as well. HypA or a homologous protein is required for nickel insertion into the hydrogenase precursor proteins. In this study, recombinant HypA from Escherichia coli was purified and characterized in vitro. Metal analysis was used to demonstrate that HypA simultaneously binds stoichiometric Zn(2+) and stoichiometric Ni(2+). Competition experiments with a metallochromic indicator reveal that HypA binds zinc with nanomolar affinity. Spectroscopic analysis of cobalt-containing HypA provides evidence for a tetrathiolate coordination sphere, suggesting that the zinc site has a structural role. In addition, HypA can exist as several oligomeric complexes and the zinc content modulates the quaternary structure of the protein. Fluorescence titration experiments demonstrate that HypA binds nickel with micromolar affinity and that the presence of zinc does not dramatically affect the nickel-binding activity. Finally, complex formation between HypA and HypB, another accessory protein required for nickel insertion, was observed. These experiments suggest that HypA is an architectural component of the hydrogenase metallocenter assembly pathway and that it may also have a direct role in the delivery of nickel to the hydrogenase large subunit.     

Purification of Rhizobium leguminosarum HypB, a nickel-binding protein required for hydrogenase synthesis.

The products of the Rhizobium leguminosarum hyp gene cluster are necessary for synthesis of a functional uptake [NiFe] hydrogenase system in symbiosis with pea plants, and at least for HypB and HypF, a role in hydrogenase-specific nickel metabolism has been postulated (L. Rey, J. Murillo, Y. Hernando, E. Hidalgo, E. Cabrera, J. Imperial, and T. Ruiz-Argüeso, Mol. Microbiol. 8:471-481, 1993). The R. leguminosarum hypB gene product has been overexpressed in Escherichia coli and purified by immobilized nickel chelate affinity chromatography in a single step. The purified recombinant HypB protein was able to bind 3.9 +/- 0.1 Ni2+ ions per HypB monomer in solution. Co2+, Cu2+, and Zn2+ ions competed with Ni2+ with increasing efficiency. Monospecific HypB antibodies were raised and used to show that HypB is synthesized in R. leguminosarum microaerobic vegetative cells and pea bacteroids but not in R. leguminosarum aerobic cells. HypB protein synthesized by R. leguminosarum microaerobic vegetative cells could also be isolated by immobilized nickel chelate affinity chromatography. A histidine-rich region at the amino terminus of the protein (23-HGHHHH DGHHDHDHDHDHHRGDHEHDDHHH-54) is proposed to play a role in nickel binding, both in solution and in chelated form.     

Dependence of Helicobacter pylori urease activity on the nickel-sequestering ability of the UreE accessory protein

The Helicobacter pylori ureE gene product was previously shown to be required for urease expression, but its characteristics and role have not been determined. The UreE protein has now been overexpressed in Escherichia coli, purified, and characterized, and three altered versions were expressed to address a nickel-sequestering role of UreE. Purified UreE formed a dimer in solution and was capable of binding one nickel ion per dimer. Introduction of an extra copy of ureE into the chromosome of mutants carrying mutations in the Ni maturation proteins HypA and HypB resulted in partial restoration of urease activity (up to 24% of the wild-type levels). Fusion proteins of UreE with increased ability to bind nickel were constructed by adding histidine-rich sequences (His-6 or His-10 to the C terminus and His-10 as a sandwich fusion) to the UreE protein. Each fusion protein was overexpressed in E. coli and purified, and its nickel-binding capacity and affinity were determined. Each construct was also expressed in wild-type H. pylori and in hypA and hypB mutant strains for determining in vivo urease activities. The urease activity was increased by introduction of all the engineered versions, with the greatest Ni-sequestering version (the His-6 version) also conferring the greatest urease activity on both the hypA and hypB mutants. The differences in urease activities were not due to differences in the amounts of urease peptides. Addition of His-6 to another expressed protein (triose phosphate isomerase) did not result in stimulation of urease, so urease activation is not related to the level of nonspecific protein-bound nickel. The results indicate a correlation between H. pylori urease activity and the nickel-sequestering ability of the UreE accessory protein.     

Ni2+ Transport and Accumulation in Rhodospirillum rubrum

The cooCTJ gene products are coexpressed with CO-dehydrogenase (CODH) and facilitate in vivo nickel insertion into CODH. A Ni(2+) transport assay was used to monitor uptake and accumulation of (63)Ni(2+) into R. rubrum and to observe the effect of mutations in the cooC, cooT, and cooJ genes on (63)Ni(2+) transport and accumulation. Cells grown either in the presence or absence of CO transported Ni(2+) with a K(m) of 19 +/- 4 microM and a V(max) of 310 +/- 22 pmol of Ni/min/mg of total protein. Insertional mutations disrupting the reading frame of the cooCTJ genes, either individually or all three genes simultaneously, transported Ni(2+) the same as wild-type cells. The nickel specificity for transport was tested by conducting the transport assay in the presence of other divalent metal ions. At a 17-fold excess Mn(2+), Mg(2+), Ca(2+), and Zn(2+) showed no inhibition of (63)Ni(2+) transport but Co(2+), Cd(2+), and Cu(2+) inhibited transport 35, 58, and 66%, respectively. Nickel transport was inhibited by cold (50% at 4 degrees C), by protonophores (carbonyl cyanide m-chlorophenylhydrazone, 44%, and 2,4-dinitrophenol, 26%), by sodium azide (25%), and hydroxyl amine (33%). Inhibitors of ATP synthase (N, N'-dicyclohexylcarbodiimide and oligomycin) and incubation of cells in the dark stimulated Ni(2+) transport. (63)Ni accumulation after 2 h was four times greater in CO-induced cells than in cells not exposed to CO. The CO-stimulated (63)Ni(2+) accumulation coincided with the appearance of CODH activity in the culture, suggesting that the (63)Ni(2+) was accumulating in CODH. The cooC, cooT, and cooJ genes are required for the increased (63)Ni(2+) accumulation observed upon CO exposure because cells containing mutations disrupting any or all of these genes accumulated (63)Ni(2+) like cells unexposed to CO.     

UreG, a chaperone in the urease assembly process, is an intrinsically unstructured GTPase that specifically binds Zn2+

Bacillus pasteurii UreG, a chaperone involved in the urease active site assembly, was overexpressed in Escherichia coli BL21(DE3) and purified to homogeneity. The identity of the recombinant protein was confirmed by SDS-PAGE, protein sequencing, and mass spectrometry. A combination of size exclusion chromatography and multiangle and dynamic laser light scattering established that BpUreG is present in solution as a dimer. Analysis of circular dichroism spectra indicated that the protein contains large portions of helices (15%) and strands (29%), whereas NMR spectroscopy indicated the presence of conformational fluxionality of the protein backbone in solution. BpUreG catalyzes the hydrolysis of GTP with a kcat=0.04 min(-1), confirming a role for this class of proteins in coupling energy requirements and nickel incorporation into the urease active site. BpUreG binds two Zn2+ ions per dimer, with a KD=42 +/- 3 microm, and has a 10-fold lower affinity for Ni2+. A structural model for BpUreG was calculated by using threading algorithms. The protein, in the fully folded state, features the typical structural architecture of GTPases, with an open beta-barrel surrounded by alpha-helices and a P-loop at the N terminus. The protein dynamic behavior observed in solution is critically discussed relative to the structural model, using algorithms for disorder predictions. The results suggest that UreG proteins belong to the class of intrinsically unstructured proteins that need the interaction with cofactors or other protein partners to perform their function. It is also proposed that metal ions such as Zn2+ could have important structural roles in the urease activation process.     

NreB from Achromobacter xylosoxidans 31A Is a nickel-induced transporter conferring nickel resistance

There are two distinct nickel resistance loci on plasmid pTOM9 from Achromobacter xylosoxidans 31A, ncc and nre. Expression of the nreB gene was specifically induced by nickel and conferred nickel resistance on both A. xylosoxidans 31A and Escherichia coli. E. coli cells expressing nreB showed reduced accumulation of Ni(2+), suggesting that NreB mediated nickel efflux. The histidine-rich C-terminal region of NreB was not essential but contributed to maximal Ni(2+) resistance.     

Metal binding sites in proteins: identification and characterization by paramagnetic NMR relaxation

A method is presented that allows the identification and quantitative characterization of metal binding sites in proteins using paramagnetic nuclear magnetic resonance spectroscopy. The method relies on the nonselective longitudinal relaxation rates of the amide protons and their dependence on the paramagnetic metal ion concentration and the pH, and on the three-dimensional structure of the protein. The method is demonstrated using Escherichia coli thioredoxin as a model protein and Ni(2+) as the paramagnetic metal ion. Through a least-squares analysis of the relaxation rates, it is found that Ni(2+) binds to a series of specific sites on the surface of thioredoxin. The strongest binding site is found near the N-terminus of the protein, where the metal ion is coordinated to the free NH(2) group of the N-terminal serine residue and the side chain carboxylate group of the aspartic acid residue in position 2. In addition, Ni(2+) binds specifically but more weakly to the surface-exposed side chain carboxylate groups of residues D10, D20, D47, and E85.