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• W2156689371 abstract "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 μm, 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 β-barrel surrounded by α-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. 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 μm, 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 β-barrel surrounded by α-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. Urease is a nickel-containing enzyme found in plants, fungi, and bacteria that catalyzes the hydrolysis of urea in the last step of nitrogen mineralization (1Ciurli S. Mangani S. Bertini I. Sigel A. Sigel H. Handbook on Metalloproteins. Marcel Dekker, Inc., New York2001: 669-708Google Scholar, 2Hausinger R.P. Karplus P.A. Messerschmidt A. Huber R. Poulos T. Wieghardt K. Handbook of Metalloproteins. John Wiley & Sons, Chichester, UK2001: 867-879Google Scholar) (Scheme 1).Scheme 1View Large Image Figure ViewerDownload Hi-res image Download (PPT) Over the past few years, intensive studies have been carried out to achieve an elucidation of its catalytic mechanism. Structures of the native enzyme isolated from Klebsiella aerogenes (Ka) 1The abbreviations used are: Ka, K. aerogenes; Bp, B. pasteurii; Hp, H. pylori; IPTG, isopropyl β-thiogalactopyranoside; DTT, dithiothreitol; ATH, alkylated thiohydantoin; MALS, multiple angle light scattering; QELS, quasi-elastic light scattering; SEC, size exclusion chromatography; TOF, time-of-flight; PDB, Protein Data Bank; ICP, induction-coupled plasma. (3Jabri E. Carr M.B. Hausinger R.P. Karplus P.A. Science. 1995; 268: 998-1004Crossref PubMed Scopus (779) Google Scholar), Bacillus pasteurii (Bp) (4Benini S. Rypniewski W.R. Wilson K.S. Miletti S. Ciurli S. Mangani S. Structure. 1999; 7: 205-216Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar), and Helicobacter pylori (Hp) (5Ha N.-C. Oh S.-T. Sung J.Y. Cha K.A. Lee M.H. Oh B.-H. Nat. Struct. Biol. 2001; 8: 505-509Crossref PubMed Scopus (388) Google Scholar) revealed a dinuclear metallo-center, with two Ni2+ ions bridged by a carbamoylated lysine residue and a hydroxide ion. The enzyme, consisting of a heterotrimeric α3β3γ3 quaternary structure with the three different subunits encoded by the ureC, ureB, and ureA genes, respectively, is synthesized in the apo-form that is devoid of nickel. The incorporation of Ni2+ into the active site, leading to the activation of the enzyme, is still poorly understood and is thought to occur in vivo as a stepwise assembly process (6Lee M.H. Mulrooney S.B. Hausinger R.P. J. Bacteriol. 1990; 172: 4427-4431Crossref PubMed Google Scholar). The assembly of the active site in vitro can be achieved by using high, nonphysiological concentrations of Ni2+ ions and bicarbonate as the source of CO2, which is needed for the carbamoylation of the active site lysine residue (7Park I.S. Hausinger R.P. Science. 1995; 267: 1156-1158Crossref PubMed Scopus (148) Google Scholar). At physiological concentrations of Ni2+ and CO2, the participation of four accessory proteins is required (8Jones B.D. Mobley H.L. J. Bacteriol. 1988; 170: 3342-3349Crossref PubMed Google Scholar, 9Mulrooney S.B. Hausinger R.P. J. Bacteriol. 1990; 172: 5837-5843Crossref PubMed Google Scholar, 10Lee M.H. Mulrooney S.B. Renner M.J. Markowicz Y. Hausinger R.P. J. Bacteriol. 1992; 174: 4324-4330Crossref PubMed Google Scholar, 11Island M.D. Mobley H.L. J. Bacteriol. 1995; 177: 5653-5660Crossref PubMed Google Scholar, 12Mobley H.L.T. Island M.D. Hausinger R.P. Microbiol. Rev. 1995; 59: 451-480Crossref PubMed Google Scholar). These proteins (UreD, UreF, UreG, and UreE) are encoded by four genes, which are also present in the urease operon together with ureA, ureB, and ureC (12Mobley H.L.T. Island M.D. Hausinger R.P. Microbiol. Rev. 1995; 59: 451-480Crossref PubMed Google Scholar). Many functional studies have been carried out on the urease accessory proteins from K. aerogenes. KaUreD (∼30 kDa) binds to apourease and appears to induce a conformational change required for the next steps of the activation process (13Park I.S. Carr M.B. Hausinger R.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3233-3237Crossref PubMed Scopus (97) Google Scholar, 14Park I.S. Hausinger R.P. Biochemistry. 1996; 35: 5345-5352Crossref PubMed Scopus (64) Google Scholar). KaUreF (∼25 kDa) binds the KaUreD-apourease complex and seems to facilitate carbamoylation of the nickel-bridging lysine residue and to prevent Ni2+ binding to the noncarbamoylated apourease (15Moncrief M.B. Hausinger R.P. J. Bacteriol. 1996; 178: 5417-5421Crossref PubMed Google Scholar). Cross-linking experiments showed an interaction between KaUreD and the α and β subunits of apourease, whereas KaUreF was shown to interact with the β subunit and to induce a conformational change capable of increasing the accessibility of the nickel ions and CO2 to residues in the active site (16Chang Z. Kuchar J. Hausinger R.P. J. Biol. Chem. 2004; 279: 15305-15313Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The interaction of UreD with UreF and with the α-subunit of apourease was suggested by immunoprecipitation and two-hybrid studies carried out on these proteins from Proteus mirabilis (17Heimer S.R. Mobley H.L. J. Bacteriol. 2001; 183: 1423-1433Crossref PubMed Scopus (33) Google Scholar). In H. pylori a similar experiment indicated the interaction between UreF and UreH, the latter corresponding to UreD in other bacteria (18Voland P. Weeks D.L. Marcus E.A. Prinz C. Sachs G. Scott D. Am. J. Physiol. 2003; 284: G96-G106Crossref PubMed Scopus (97) Google Scholar). KaUreG (∼22 kDa) can form a quaternary complex with KaUreDF-apourease, suggesting that such large aggregate is the minimum competent species for the in vitro urease activation (7Park I.S. Hausinger R.P. Science. 1995; 267: 1156-1158Crossref PubMed Scopus (148) Google Scholar, 19Park I.S. Hausinger R.P. J. Bacteriol. 1995; 177: 1947-1951Crossref PubMed Google Scholar) and could be required for the process occurring in vivo (20Moncrief M.B. Hausinger R.P. J. Bacteriol. 1997; 179: 4081-4086Crossref PubMed Scopus (98) Google Scholar). Finally, KaUreE (a homodimer of ∼35 kDa) is thought to bind the KaUreDFG-apourease complex, acting as a nickel transporter that delivers Ni2+ to the active site of the enzyme (21Lee M.H. Pankratz H.S. Wang S. Scott R.A. Finnegan M.G. Johnson M.K. Ippolito J.A. Christianson D.W. Hausinger R.P. Protein Sci. 1993; 2: 1042-1052Crossref PubMed Scopus (124) Google Scholar, 22Brayman T.G. Hausinger R.P. J. Bacteriol. 1996; 178: 5410-5416Crossref PubMed Google Scholar, 23Colpas G.J. Brayman T.G. McCracken J. Pressler M.A. Babcock G.T. Ming L-J. Colangelo C.M. Scott R.A. Hausinger R.P. J. Biol. Inorg. Chem. 1998; 3: 150-160Crossref Scopus (37) Google Scholar, 24Colpas G.J. Brayman T.G. Ming L.J. Hausinger R.P. Biochemistry. 1999; 38: 4078-4088Crossref PubMed Scopus (81) Google Scholar, 25Colpas G.J. Hausinger R.P. J. Biol. Chem. 2000; 275: 10731-10737Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Among the four accessory proteins, UreG plays an essential role in coupling cellular metabolism and bioenergetics to the assembly of urease. This protein contains a fully conserved P-loop motif, which is also present in many nucleotide-binding proteins and which is probably related to the in vivo GTP requirement for assembly of the urease active site (26Soriano A. Hausinger R.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11140-11144Crossref PubMed Scopus (102) Google Scholar). A direct relationship between UreG and GTP requirement is proven by the evidence that GTP is needed for activation of the Ka-UreDFG-apourease complex, although it has an inhibitory effect on the nickel reconstitution of the apourease and of the KaUreD-apourease and KaUreDF-apourease complexes (26Soriano A. Hausinger R.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11140-11144Crossref PubMed Scopus (102) Google Scholar). UreG is also involved in delivering CO2 necessary for the carbamoylation of the nickel-bridging lysine; the curves that correlate the urease activation to different bicarbonate concentrations indicate a higher rate and level of enzymatic activation in the presence of UreDFG-apourease complex (26Soriano A. Hausinger R.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11140-11144Crossref PubMed Scopus (102) Google Scholar). In vitro, optimal levels of apourease activation require 0.5 mm GTP. Larger GTP concentrations lead to a decrease of urease activity, probably caused by the chelation of Ni2+ by the nucleotide; this is consistent with the fact that elevated levels of Mg2+ ions partially restore the activation (26Soriano A. Hausinger R.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11140-11144Crossref PubMed Scopus (102) Google Scholar). In the presence of UreE, this inhibitory effect is lost, and the urease activation occurs at significantly lower GTP concentrations (27Soriano A. Colpas G.J. Hausinger R.P. Biochemistry. 2000; 39: 12435-12440Crossref PubMed Scopus (83) Google Scholar). These observations indicate a correlation between GTP hydrolysis by UreG and Ni2+ transfer to apourease by UreE. Additional evidence of the UreE-UreG interaction in vivo has been obtained by using two-hybrid systems and immunoprecipitation experiments in H. pylori (18Voland P. Weeks D.L. Marcus E.A. Prinz C. Sachs G. Scott D. Am. J. Physiol. 2003; 284: G96-G106Crossref PubMed Scopus (97) Google Scholar). It has been proposed that UreG may induce GTP-dependent structural changes of the apourease, increasing the accessibility of both Ni2+ and CO2 to the developing active site. Alternatively, UreG may use GTP and CO2 to synthesize carboxyphosphate, which could serve as an excellent CO2 donor to the Lys residue (26Soriano A. Hausinger R.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11140-11144Crossref PubMed Scopus (102) Google Scholar). The thorough functional studies described above have paved the way to an understanding of the mechanism of the urease accessory proteins and, in particular, of UreG in Ni2+ trafficking and metabolic regulation. The next echelon in the comprehension of the urease chemistry implies the study of the interaction mechanisms between the accessory proteins and the enzyme at the molecular level, in order to understand how the urease active site is assembled. This goal cannot be achieved without detailed structural information on each accessory protein and its biochemical properties. The only crystal structures available for the urease chaperones are those of UreE from K. aerogenes (28Song H-K. Mulrooney S.B. Huber R. Hausinger R.P. J. Biol. Chem. 2001; 276: 49359-49364Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) and B. pasteurii (29Remaut H. Safarov N. Ciurli S. Van Beeumen J.J. J. Biol. Chem. 2001; 276: 49365-49370Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The high degree of similarity between these two structures and between the structures of Ka- and Bp-urease are indications of a conserved molecular mechanism of urease activity, activation, and metal-center building in different species (30Musiani F. Zambelli B. Stola M. Ciurli S. J. Inorg. Biochem. 2004; 98: 803-813Crossref PubMed Scopus (38) Google Scholar). No structural detail is available for the other three chaperones. A study, published in 1997 (20Moncrief M.B. Hausinger R.P. J. Bacteriol. 1997; 179: 4081-4086Crossref PubMed Scopus (98) Google Scholar), reported that KaUreG is monomeric in solution and that it does not, by itself, hydrolyze GTP or ATP. Another report in 2003 (31Mehta N. Benoit S. Maier R.J. Microb. Pathog. 2003; 35: 229-234Crossref PubMed Scopus (62) Google Scholar) and concerning HpUreG essentially confirmed these results. This paper describes a thorough study performed on the recombinant UreG from B. pasteurii. In particular, the cloning, expression, and purification of the protein in its native and His-tagged forms are described, together with evidence confirming the identity of the isolated protein. The oligomerization state and the hydrodynamic properties of the protein in solution were examined by using size exclusion chromatography coupled with light scattering experiments, and the protein folding was checked using circular dichroism, mass spectrometry, and NMR. The metal-binding capability and enzymatic GTPase activity of BpUreG were established for the first time and discussed. Finally, threading (fold recognition) algorithms were applied to calculate a model for the protein structure that is consistent with its solution properties and enzymatic activity. The results represent a significant contribution to the understanding of the role of this metallochaperone in the urease active site assembly. BpUreG Cloning—In accordance with the DNA sequence of the B. pasteurii urease operon available from GenBank™ (accession number AF361945), two 24- and 26-bp oligonucleotide primers were designed and synthesized to amplify the ureG gene by the PCR technique using the pUC19 plasmid containing the B. pasteurii ure operon (32Ciurli S. Safarov N. Miletti S. Dikiy A. Christensen S.K. Kornetzky K. Bryant D.A. Vandenberghe I. Devreese B. Samyn B. Remaut H. Van Beeumen J.J. J. Biol. Inorg. Chem. 2002; 7: 623-631Crossref PubMed Scopus (41) Google Scholar) as template. The following forward and reverse primers introduced NdeI and BamHI restriction enzyme recognition sites, respectively (boldface type): 5′-CTAGGAGATTGTGCATATGAAAAC-3′; 5′-CAATATCGAG GGATCCAAACGGTATT-3′. Taq polymerase for the PCR and dNTPs were from Display System Biotech. The oligonucleotide primers were synthesized in the Nucleic Acid Facility at the Pennsylvania State University (University Park, PA). The PCR product obtained by using these primers was digested by a combination of NdeI and BamHI restriction enzymes (New England Biolabs), purified by electrophoresis on a 1% (w/v) agarose gel, extracted, and precipitated. By using T4 DNA ligase (Promega), this DNA fragment was ligated at a 2-fold excess of insert to vector, into plasmid pET3a (Novagen), which had been digested with NdeI and BamHI, treated with alkaline phosphatase, and purified by electrophoresis. For the His-tagged BpUreG, the plasmid pET15b was used. Plasmid DNA was isolated from transformants of Escherichia coli strain DH5α (Invitrogen) by the rapid alkaline extraction method, as described (33Sambrook J. Fritisch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar), digested with appropriate restriction enzymes, and analyzed by agarose gel electrophoresis. The resulting pET3a::ureG and pET15b::ureG plasmids were purified using the StrataPrep™ Plasmid MiniPrep kit (Stratagene). The sequence of the cloned BpUreG gene was confirmed by DNA sequencing. The constructs for both plasmids were inserted by electroporation (Bio-Rad GenePulser II) into the E. coli BL21(DE3) expression host (Novagen) grown in shaking flasks at 37 °C in a medium with the Luria-Bertani (LB) composition (Amersham Biosciences) or on agar (1.5%) plates with the same composition. BpUreG Expression—Based on the T7 system (34Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 66-89Google Scholar), large scale expression of BpUreG and His-tagged BpUreG was achieved in 2.5-liter batches of minimum M9 liquid media (1 liter contained6gofNa2HPO4, 3 g of KH2PO4, 0.5 g of NaCl, 1.25 g of (NH4)2SO4, 0.246 g of MgSO4) supplemented with 4 g of glucose per liter of culture. The 15N-enriched proteins were obtained using a medium containing (15NH4)2SO4. Transformed E. coli BL21(DE3) cells were grown at 37 °C (28 °C for the His-tagged protein) with vigorous stirring, until the A600 reached 0.5–0.8. Expression was induced by addition of isopropyl β-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mm. The cells were harvested 4 h after induction by centrifugation at 8,000 × g for 10 min at 4 °C. The cells were resuspended in 25 ml of 50 mm Tris-HCl buffer, pH 8, containing 5 mm EDTA and lysozyme (200 μg/ml). After incubation at 30 °C for 20 min, followed by the addition of DNase I (20 μg ml-1) and additional incubation at 37 °C for 20 min, the cells were disrupted by two passages through a French pressure cell (SLM, Aminco) at 20,000 pounds/square inch. The cell pellet was separated from the supernatant by centrifugation at 15,000 × g for 15 min at 4 °C. BpUreG Purification—In the case of native BpUreG, the pellet was washed (resuspended in 25 ml of buffer using a mixer homogenizer and centrifuged at 15,000 × g for 15 min at 4 °C) three times with 50 mm Tris-HCl buffer, pH 8, containing 5 mm EDTA, 1 mm DTT, and 2% (w/v) Triton X-100 and three times with the same buffer without Triton X-100. The pellet was subsequently resuspended and incubated overnight at 4 °C in 50 mm Tris-HCl buffer, pH 8, containing 1 mm DTT and 2 m urea. The soluble fraction, obtained after removal of the precipitated material by centrifugation (15,000 × g, 15 min), was loaded onto a Q-Sepharose XK 26/10 column (Amersham Biosciences) that had been pre-equilibrated with 2 volumes of 50 mm Tris-HCl buffer, pH 8, containing 1 mm DTT and 2 m urea. The column was washed using a flow rate of 3 ml min-1 with the starting buffer until the base line was stable. The protein was eluted from the column with a 400-ml linear gradient of NaCl (0–1 m). Fractions containing BpUreG were combined, diluted with the elution buffer to a protein concentration of 0.3 mg/ml, and dialyzed (5-kDa cut-off membrane) overnight at 4 °C against 50 mm Tris-HCl buffer, pH 8. The resulting solution of BpUreG was concentrated by using 5-kDa cut-off membrane Amicon and Centricon ultrafiltration units (Millipore), to a final volume of 5 ml, and centrifuged (15 min at 14,000 × g) to remove the precipitated material. The resulting solution was loaded onto a Superdex 75 XK 26/60 column conditioned with 50 mm Tris-HCl buffer, pH 8, containing 0.15 m NaCl and 1 mm DTT. BpUreG was eluted at a flow rate of 2 ml min-1, and the purified protein, amounting to ∼50 mg per liter of culture, was concentrated to 2.5 mg ml-1 and stored at -80 °C. In the case of His-tagged BpUreG, the supernatant after pellet separation was loaded onto a column containing 8 ml of the nickel-nitrilotriacetic acid Superflow affinity resin (Qiagen) pre-equilibrated with 40 ml of 50 mm Tris-HCl buffer, pH 8.0, containing 5 mm imidazole, washed with 30 ml of the same buffer containing 20 mm imidazole, and eluted using the same buffer containing 100 mm imidazole. Protein purity, as well as the molecular mass of BpUreG in denaturing conditions, was estimated by SDS-PAGE according to the method of Laemmli (35Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar), by using a Bio-Rad Mini-Protean II apparatus. Proteins were separated on 15% (w/v) acrylamide-bisacrylamide separating gels that were stained using either Coomassie Brilliant Blue R-250 or silver staining. Protein concentration was measured by using a Jasco 7800 spectro-photometer and a value for the extinction coefficient (ϵ280 = 10,810 m-1 cm-1) calculated from the amino acid sequence using the ProtParam web site (au.expasy.org/tools/protparam.html). This value is in good agreement with that obtained by using the Bio-Rad assay that is based on the Bradford colorimetric method (36Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Protein Sequence Determination—All reagents, solvents and instruments were obtained from Applied Biosystems. N-terminal sequence analysis was performed in the gas-pulsed liquid phase using a model 476A protein sequencer with a micro-reaction chamber and an on-line high pressure liquid chromatography system for phenylthiohydantoin analysis. Absorbance was monitored at 269 nm. C-terminal sequence analysis was performed on a Procise 494C protein sequencer using C-terminal sequencing chemistry (37Boyd V.L. Bozzini M. Zon G. Noble R.L. Mattaliano R.J. Anal. Biochem. 1992; 206: 344-352Crossref PubMed Scopus (79) Google Scholar, 38Samyn B. Van der Hardeman K. Eycken J. Van Beeumen J. Anal. Chem. 2000; 72: 1389-1399Crossref PubMed Scopus (31) Google Scholar). Prior to this analysis, the sample was adsorbed on a Prosorb sample preparation cartridge and, after subsequent washes with MilliQ-filtered water, treated with 200 mm phenylisocyanate in acetonitrile under basic conditions (124 mm diisopropylethylamine/acetonitrile) in order to modify the ϵ-amino group of the lysine residues into stable phenylureas. The alkylated thiohydantoin (ATH)-amino acids were analyzed on-line using a thermostated (38 °C) C18 reverse-phase column (2.1 × 220 mm, 5 μm). A linear gradient with a flow rate of 300 μl min-1 was formed using a 140C microgradient system with the following solvents: solvent A, 35 mm sodium acetate buffer, 3.5% tetrahydrofuran/MQ water, and solvent B, 100% acetonitrile. The ATH-amino acid derivatives were monitored using a 785A absorbance detector set at 254 nm, and quantified relative to a 100 pmol of ATH-amino acid standard. The methyl naphthylthiohydantoin amino acid standards were obtained from the supplier. Mass Spectrometry—All mass spectrometric analyses were performed on a quadrupole TOF mass spectrometer (Micromass), interfaced to a chip-based nano-ESI source (NanoMate100, Advion Biosciences). The mass spectra were processed using MassLynx version 3.1 software of Micromass. Before analysis, the buffer was changed to 50 mm NH4 acetate, pH 6.5. The denatured protein (1 μm) was measured in 50% acetonitrile, 0.1% formic acid. For native measurements, the protein (5 μm) was kept in 50 mm NH4 acetate, pH 6.5. Acquisition conditions were a spraying voltage of 1.5 (denatured protein) or 1.7 kV (native protein), gas pressure of 0.3 pounds/square inch, and an acquisition time of 3 (denatured protein) or 10 min (native protein) across an m/z range of 500–3000. Hydrodynamic Properties—The molecular mass and hydrodynamic radius of the native protein were estimated by standard size exclusion chromatography. A small amount (100 μl, 2.5 mg/ml) of the purified protein solution was applied to a Superdex-75 HR 10/30 FPLC column that had been equilibrated with 50 mm Tris-HCl, pH 8.0, containing 0.15 m NaCl, at a flow rate of 0.5 ml min-1 in order to estimate the apparent hydrodynamic volume of the purified protein. The column was calibrated using an Amersham Biosciences low molecular weight gel filtration calibration kit. Absolute estimates of molecular mass and hydrodynamic radius of BpUreG were determined using a combination of size exclusion chromatography, multiple angle light scattering (MALS), and quasi-elastic light scattering (QELS). BpUreG (100 μl, 2.5 mg/ml) in 20 mm Tris-HCl, pH 7.5, 150 mm NaCl was loaded onto an S-200 16/60 column (Amersham Biosciences), pre-equilibrated with the same buffer, and eluted at room temperature at a flow rate of 1 ml/min. The column was connected downstream to a multiangle laser light (690.0 nm) scattering DAWN EOS photometer (Wyatt Technology). Quasi-elastic (dynamic) light scattering data were collected at a 90° angle by using a Wyatt-QELS device. The concentration of the eluted protein was determined using a refractive index detector (Optilab DSP, Wyatt). Values of 0.185 for the refractive index increment (dn/dc) and 1.330 for the solvent refractive index were used. Molecular weights were determined from a Zimm plot. Data were analyzed using the Astra 4.90.07 software (Wyatt Technology), following the manufacturer's indications. Circular Dichroism Spectroscopy—The CD spectra of BpUreG and its His-tagged analogue were measured at 20 °C, using a Jasco 710 spectropolarimeter flushed with N2, and a cuvette with 0.01-cm path length. The buffer was 20 mm phosphate, pH 7.5, containing 0.15 m NaCl. The spectra were registered from 190 to 300 nm at 0.2-nm intervals. Ten spectra were accumulated at room temperature and averaged to achieve an appropriate signal-to-noise ratio. The spectrum of the buffer was subtracted. The secondary structure composition of BpUreG was evaluated with the tool available on the Dichroweb server of the Centre for Protein and Membrane Structure and Dynamics, www.cryst.bbk.ac.uk/cdweb/html/home.html (39Lobley A. Whitmore L. Wallace B.A. Bioinformatics. 2002; 18: 211-212Crossref PubMed Scopus (645) Google Scholar) using the reference sets 4 and 7. NMR Spectroscopy—NMR spectra of 15N-enriched KaUreG and His-tagged BpUreG were recorded at pH 8.0 and 298 K on a Bruker Avance 800 spectrometer operating at 800.13 MHz. The KaUreG spectrum was recorded using a 5-mm reverse detection probe on a 1-mM sample, whereas the spectrum of His-tagged BpUreG was obtained using a TXI cryoprobe on a 0.45-mM sample. The spectrum of BpUreG isolated from inclusion bodies was recorded on a 0.45-mm 15N-enriched sample at pH 8.0 and 298 K using a Bruker DRX Avance 500 spectrometer operating at 500.13 MHz and equipped with a TXO cryoprobe. 1H,15N-HSQC spectra were acquired using sensitivity improvement (40Palmer A.G.I. Cavanagh J. Wright P.E. Rance M. J. Magn. Reson. 1991; 93: 151-170Google Scholar, 41Kay L.E. Keifer P. Saarinen T. J. Am. Chem. Soc. 1992; 114: 10663-10665Crossref Scopus (2439) Google Scholar, 42Schleucher J. Schwendinger M. Sattler M. Schmidt P. Schedletzky O. Glaser S.J. Sorensen O.W. Griesinger C. J. Biomol. NMR. 1994; 4: 301-306Crossref PubMed Scopus (720) Google Scholar) and consisted of 8–48 scans, spectral windows of 11–16 ppm in the proton dimension, and 30–40 ppm in the nitrogen dimension, with the carrier set at the water frequency and 118 ppm, respectively. Relaxation delays (including acquisition time) in the range 0.9–1.2 s were employed. Matrices of 1024 × 256 points or 2048 × 128 points were acquired and transformed into 1024 × 512 or 2048 × 512 points. Measurement of GTPase Activity—GTP hydrolyzing activity was measured using a colorimetric method. The reaction mixture, containing 20 mm Tris-HCl, pH 8.0, 0.075 m NaCl, 5 mm MgCl2,2mm GTP, and 5 μm BpUreG, in the absence or presence of 25 μm ZnSO4, was incubated at 37 °C. Aliquots (90 μl) were removed at different incubation times and added to 30 μl of a 35% trichloroacetic acid/water solution. Phosphate concentration was determined by the malachite green assay (43Lanzetta P.A. Alvarez L.J. Reinach P.S. Candia O.A. Anal. Biochem. 1979; 100: 95-97Crossref PubMed Scopus (1821) Google Scholar). Metal Binding Experiments—In all operations, care was taken to avoid exogenous metal contamination. Ni2+ and Zn2+ nitrate salts solutions were prepared starting from ICP 1000 ppm standard solutions (CPI International) diluted to 1 mm with buffer A, containing NaCl 0.15 m. 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• W2156689371 creator A5059566217 @default.
• W2156689371 date "2005-02-01" @default.
• W2156689371 modified "2023-10-03" @default.
• W2156689371 title "UreG, a Chaperone in the Urease Assembly Process, Is an Intrinsically Unstructured GTPase That Specifically Binds Zn2+" @default.
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