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• W2094139743 abstract "Genetic evidence has indicated that Isc proteins play an important role in iron-sulfur cluster biogenesis. In particular, IscU is believed to serve as a scaffold for the assembly of a nascent iron-sulfur cluster that is subsequently delivered to target iron-sulfur apoproteins. We report the characterization of an IscU fromThermatoga maritima, an evolutionarily ancient hyperthermophilic bacterium. The stabilizing influence of a D40A substitution allowed characterization of the holoprotein. Mössbauer (δ = 0.29 ± 0.03 mm/s, ΔEQ = 0.58 ± 0.03 mm/s), UV-visible absorption, and circular dichroism studies of the D40A protein show that T. maritima IscU coordinates a [2Fe-2S]2+ cluster. Thermal denaturation experiments demonstrate that T. maritima IscU is a thermally stable protein with a thermally unstable cluster. This is also the first IscU type domain that is demonstrated to possess a high degree of secondary and tertiary structure. CD spectra indicate 36.7% α-helix, 13.1% antiparallel β-sheet, 11.3% parallel β-sheet, 20.2% β-turn, and 19.1% other at 20 °C, with negligible spectral change observed at 70 °C. Cluster coordination also has no effect on the secondary structure of the protein. The dispersion of signals in1H-15N heteronuclear single quantum correlation NMR spectra of wild type and D40A IscU supports the presence of significant tertiary structure for the apoprotein, consistent with a scaffolding role, and is in marked contrast to other low molecular weight Fe-S proteins where cofactor coordination is found to be necessary for proper protein folding. Consistent with the observed sequence homology and proposed conservation of function for IscU-type proteins, we demonstrate T. maritimaIscU-mediated reconstitution of human apoferredoxin. Genetic evidence has indicated that Isc proteins play an important role in iron-sulfur cluster biogenesis. In particular, IscU is believed to serve as a scaffold for the assembly of a nascent iron-sulfur cluster that is subsequently delivered to target iron-sulfur apoproteins. We report the characterization of an IscU fromThermatoga maritima, an evolutionarily ancient hyperthermophilic bacterium. The stabilizing influence of a D40A substitution allowed characterization of the holoprotein. Mössbauer (δ = 0.29 ± 0.03 mm/s, ΔEQ = 0.58 ± 0.03 mm/s), UV-visible absorption, and circular dichroism studies of the D40A protein show that T. maritima IscU coordinates a [2Fe-2S]2+ cluster. Thermal denaturation experiments demonstrate that T. maritima IscU is a thermally stable protein with a thermally unstable cluster. This is also the first IscU type domain that is demonstrated to possess a high degree of secondary and tertiary structure. CD spectra indicate 36.7% α-helix, 13.1% antiparallel β-sheet, 11.3% parallel β-sheet, 20.2% β-turn, and 19.1% other at 20 °C, with negligible spectral change observed at 70 °C. Cluster coordination also has no effect on the secondary structure of the protein. The dispersion of signals in1H-15N heteronuclear single quantum correlation NMR spectra of wild type and D40A IscU supports the presence of significant tertiary structure for the apoprotein, consistent with a scaffolding role, and is in marked contrast to other low molecular weight Fe-S proteins where cofactor coordination is found to be necessary for proper protein folding. Consistent with the observed sequence homology and proposed conservation of function for IscU-type proteins, we demonstrate T. maritimaIscU-mediated reconstitution of human apoferredoxin. nitrogen fixation heteronuclear single quantum correlation dithiothreitol electron paramagnetic resonance electrospray ionization ferredoxin human ferredoxin iron-sulfur cluster wild type nickel-nitrilotriacetic acid high performance liquid chromatography Iron-sulfur (Fe-S) cluster proteins participate in a wide variety of physiologically essential processes including gene regulation, electron transfer, and catalytic reactions (1Beinert H. J. Biol. Inorg. Chem. 2000; 5: 2-15Crossref PubMed Scopus (535) Google Scholar). In vitro it is possible to reconstitute Fe-S apoproteins by anaerobic incubation with iron, sulfide, and a suitable reducing agent. However, because of the cellular toxicity of free iron and sulfide, it is believed that Fe-S cluster biogenesis is mediated by specific protein-protein interactions rather than by spontaneous formation. Initial identification of essential Fe-S cluster maturation components was made within thenif1 operon of Azotobacter vinelandii. Disruption of eithernifS or nifU resulted in the loss of Fe-S coordination to nitrogenase (2Jacobson M.R. Cash V.L. Weiss M.C. Laird N.F. Newton W.E. Dean D.R. Mol. Gen. Genet. 1989; 219: 45-57Crossref Scopus (247) Google Scholar). NifS has been shown to provide sulfur equivalents by catalytic cysteine desulfurization (3Zheng L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (503) Google Scholar); however, the role of NifU has yet to be firmly established. NifU is a modular protein with three domains. The amino-terminal domain has three conserved Cys and binds a labile less stable [2Fe-2S] cluster, the central region contains four conserved Cys and coordinates a stable [2Fe-2S] cluster, and the carboxyl-terminal domain has an unknown function with two conserved Cys (4Agar J.N. Krebs C. Frazzon J. Huynh B.H. Dean D.R. Johnson M.K. Biochemistry. 2000; 39: 7856-7862Crossref PubMed Scopus (386) Google Scholar, 5Fu W. Jack R.F. Morgan T.V. Dean D.R. Johnson M.K. Biochemistry. 1994; 33: 13455-13463Crossref PubMed Scopus (129) Google Scholar, 6Hwang D.M. Dempsey A. Tan K.-T. Liew C.-C. J. Mol. Evol. 1996; 43: 536-540Crossref PubMed Scopus (52) Google Scholar). Of these domains the central region has been the most thoroughly characterized. Through DNA sequencing it has been observed that nif homologues are encoded within the genomes of most organisms. These homologues have been named isc (for iron sulfurcluster) and are usually clustered together, encoding IscS, IscU, IscA, heat shock proteins HscB and HscA, and a ferredoxin (7Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). The NifU homologue, IscU, is only homologous to the amino terminus of NifU and analogously coordinates a reductively labile [2Fe-2S] cluster (6Hwang D.M. Dempsey A. Tan K.-T. Liew C.-C. J. Mol. Evol. 1996; 43: 536-540Crossref PubMed Scopus (52) Google Scholar, 7Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar, 8Foster M.W. Mansy S.S. Hwang J. Penner-Hahn J.E. Surerus K.K. Cowan J.A. J. Am. Chem. Soc. 2000; 122: 6805-6806Crossref Scopus (78) Google Scholar, 9Yuvaniyama P. Agar J.N. Cash V.L. Johnson M.K. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 599-604Crossref PubMed Scopus (277) Google Scholar). In eukaryotes, Isc proteins have been identified in mitochondria (10Lill R. Kispal G. Trends Biochem. Sci. 2000; 25: 352-356Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Results from genetic screens of A. vinelandii, in vitro characterization of NifU/IscU chemistry, and the high degree of conservation of NifU-like proteins from divergent species have led to the hypothesis that NifU/IscU proteins, in conjunction with NifS/IscS, deliver Fe-S cluster equivalents to target Fe-S apoproteins (4Agar J.N. Krebs C. Frazzon J. Huynh B.H. Dean D.R. Johnson M.K. Biochemistry. 2000; 39: 7856-7862Crossref PubMed Scopus (386) Google Scholar). Thermatoga maritima is a hyperthermophilic bacterium. It represents one of the deepest and most slowly evolving eubacterial lineages (11Bocchetta M. Gribaldo S. Sanangelantoni A. Cammarano P. J. Mol. Evol. 2000; 50: 366-380Crossref PubMed Scopus (67) Google Scholar), with an optimal growth temperature of 80 °C (12Stetter K.O. FEMS Microbiol. Rev. 1996; 18: 149-158Crossref Google Scholar). The genomic sequence shows genes encoding both IscU (Tm IscU) and IscS located next to each other, although no other iscgenes are found clustered within this region. Located elsewhere in theT. maritima genome are heat shock proteins that are homologous to those identified in other organisms (13Michelini E.T. Flynn G.C. J. Bacteriol. 1999; 181: 4237-4244Crossref PubMed Google Scholar) and several ferredoxins. Additionally, T. maritima expresses a second NifS-like protein that has been crystallographically characterized (14Kaiser J.T. Clausen T. Bourenkow G.P. Bartunik H.-D. Steinbacher S. Huber R. J. Mol. Biol. 2000; 297: 451-464Crossref PubMed Scopus (126) Google Scholar). Possibly, the functions of the missing Isc proteins are provided by other unidentified proteins involved in iron homeostasis, as has been identified in Escherichia coli (15Muller K. Matzanke B.F. Schunemann V. Trautwein A.X. Hantke K. Eur. J. Biochem. 1998; 258: 1001-1008Crossref PubMed Scopus (48) Google Scholar, 16Patzer S.I. Hantke K. J. Bacteriol. 1999; 181: 3307-3309Crossref PubMed Google Scholar). Herein we report the characterization of an evolutionarily ancient IscU. Similar to other bacterial and eukaryotic organisms (8Foster M.W. Mansy S.S. Hwang J. Penner-Hahn J.E. Surerus K.K. Cowan J.A. J. Am. Chem. Soc. 2000; 122: 6805-6806Crossref Scopus (78) Google Scholar, 17Agar J.N. Zheng L. Cash V.L. Dean D.R. Johnson M.K. J. Am. Chem. Soc. 2000; 122: 2136-2137Crossref Scopus (116) Google Scholar,18Urbina H.D. Silberg J.J. Hoff K.G. Vickery L.E. J. Biol. Chem. 2001; 276: 44521-44526Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar), Tm IscU binds a [2Fe-2S] cluster. The holo form of the native protein proved to be relatively labile, but following prior precedent (8Foster M.W. Mansy S.S. Hwang J. Penner-Hahn J.E. Surerus K.K. Cowan J.A. J. Am. Chem. Soc. 2000; 122: 6805-6806Crossref Scopus (78) Google Scholar, 9Yuvaniyama P. Agar J.N. Cash V.L. Johnson M.K. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 599-604Crossref PubMed Scopus (277) Google Scholar) was stabilized by substitution of a conserved aspartate by alanine (D40A). Factors that promote stabilization through this substitution will be described elsewhere. Analysis of CD spectra over a wide temperature range demonstrate Tm IscU to possess significant secondary structure that is not dependent upon the coordination of an Fe-S cluster, whereas the dispersion of resonances in 1H-15N HSQC spectra indicate significant tertiary structure. Evidence for secondary and tertiary structure was absent for the human and yeast homologues that we have examined in our laboratory, and reasons for this variation are discussed. Furthermore, Tm IscU proves to be competent for Fe-S cluster transfer to human ferredoxin (Hs Fd), consistent with a conserved role in Fe-S cluster biogenesis. This result provides strong support for a common conserved recognition mechanism for both prokaryotic and eukaryotic IscU-type proteins. 57Fe was from Pennwood Chemicals. All restriction enzymes and the copper staining kit were from Invitrogen. Pfu DNA polymerase and BL21CodonPlus(DE3)-RIL were from Stratagene (La Jolla, CA); PCR purification kit and Ni-NTA resin were purchased from Qiagen (Valencia, CA). Protein expression vectors pET21, pET28, and BL21(DE3) pLysS were from Novagen (Madison, WI). Oligonucleotides were from Integrated DNA Technologies, Inc. (Coralville, IA). CM-32 and DE-52 were from Whatman (Aston, PA). Homogenous-20 precast polyacrylamide gels and G-75 and Superose-12 resins were obtained from Amersham Biosciences. T. maritima genomic DNA was obtained from the American Type Culture Collection (ATCC no. 43589D). DNA (50 ng), 0.2 μm amounts of each primer, 2.5 units of clonedPfu DNA polymerase, 1× cloned Pfu buffer, and 0.2 mm each dNTP were used to amplifyiscU (TIGR locus TM1372, www.tigr.org) via PCR. Primers were: 5′-GGGCCCGGCATATGGTTTTCAAGATGATG-3′ and 5′-CCGGCCGGATCCTTAAGGCCGTGAAATCTTTTTG-3′, where underlined regions denote NdeI and BamHI sites, respectively, and the bold position indicates a G to A substitution resulting in an amino-terminal Met rather than a Val for improved recombinant expression in E. coli. The thermocycle used was identical to that described in the Pfu DNA polymerase manual (Stratagene). PCR products were digested with NdeI and BamHI and ligated to similarly treated pET21 and pET28. Cloning into pET21 yielded a construct without a tag or additional residues (pTmIscU). Cloning into pET28 resulted in the addition of an amino-terminal His tag (pTmIscUHis). The QuikChange technique (Stratagene) was employed for the D40A point mutation. Reactions contained 50 ng of template (either pTmIscU or pTmIscUHis), 2.5 units of cloned Pfu DNA polymerase, 1× cloned Pfu buffer, 0.5 mm DTT, and 125 ng of each primer. Primers were 5′-GGGAAAGAACATCTCTTGTGGCGCCGAAATCACACTCTAC-3′ and 5′-GTAGAGTGTGATTTCGGCGCCACAAGAGATGTTCTTTCCC-3′, where the bold positions indicate the mutation. The thermocycle was identical to that described in the QuikChange manual (Stratagene). An aliquot (27%) of the post-thermocycle sample was incubated with 7.5 units ofDpnI at 37 °C for 2 h. Subsequently, CaCl2-competent DH5α was transformed via heat shock with the mutant constructs (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Plainview, NY1989Google Scholar). Cloning and mutagenesis results were confirmed by nucleotide sequencing at the Ohio State University Plant-Microbe Genomics Facility. BL21CodonPlus(DE3)-RIL was used for protein expression. A 100-ml Luria-Bertani culture (supplemented with either 100 μg/ml ampicillin and 35 μg/ml chloramphenicol for pTmIscU constructs or 50 μg/ml kanamycin and 35 μg/ml chloramphenicol for pTmIscUHis constructs) was grown overnight as a starter culture. The entire starter culture was used as an inoculum for a 10-liter fermentation at the Ohio State University fermentation facility and grown to anA600 ∼ 0.6 prior to induction with 1 mm isopropyl-1-thio-β-d-galactopyranoside. Cells were pelleted 5 h after induction and stored at –80 °C for future use. Cell pellets were resuspended in five volumes of 50 mm Tris-HCl, pH 7.4, 2 mmβ-mercaptoethanol, 20 μg/ml DNase, and 5 μg/ml RNase and lysed by sonication. Insoluble material was removed by centrifugation at 15,000 rpm, 4 °C for 1 h. For His-tagged constructs, the cleared lysate was applied to a Ni-NTA column equilibrated with binding buffer (20 mm Tris-HCl, pH 7.9, 5 mm imidazole, 500 mm NaCl). The column was then washed with five column volumes of binding buffer and five volumes of binding buffer + 15 mm imidazole, and the protein was subsequently eluted with binding buffer + 100 mm imidazole. His-tagged protein was exchanged with 50 mm sodium phosphate, pH 7.4, via repeated ultrafiltration (Amicon). The cleared lysate containing non-His-tagged protein was loaded onto a cation exchange column (CM32) equilibrated with 50 mmsodium phosphate, pH 7.4, and washed with one cleared lysate volume of phosphate buffer. The flow-through and wash fractions were combined. NaCl was added to 50 mm, β-mercaptoethanol was added to 5 mm, and the solution was incubated at 85 °C for 0.5 h. The sample was then centrifuged at 15,000 rpm, 4 °C for 10 min and the supernatant loaded onto an anion exchange column (DE-52). The column was washed with three column volumes of 50 mmTris-HCl, pH 7.4. The flow-through and wash fractions were combined and concentrated via ultrafiltration. Subsequently, protein solution was loaded onto a G-75 gel filtration column equilibrated with 50 mm sodium phosphate, pH 7.4. The fractions with a λmax at 278 nm were pooled and confirmed to be pure IscU via SDS-PAGE. All apoprotein samples were stored at either 4 or –80 °C. HuFd/pET3a (encoding human ferredoxin) was a gift from J. L. Markley. BL21(DE3) pLysS HuFd/pET3a was essentially expressed and purified as previously described (20Xia B. Cheng H. Bandarian V. Reed G.H. Markley J.L. Biochemistry. 1996; 35: 9488-9495Crossref PubMed Scopus (76) Google Scholar). Hs apoFd was prepared as described by Nishio and Nakai (21Nishio K. Nakai M. J. Biol. Chem. 2000; 275: 22615-22618Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). All mass spectra were acquired at the campus chemical instrument center at Ohio State University, and all solutions were made in Barnstead purified water. Molecular mass determination for extinction coefficient calculation was determined by electrospray ionization (ESI) using a Micromass Q-TOF™ II (Micromass, Wythenshawe, UK) mass spectrometer equipped with an orthogonal electrospray source (Z-spray) operated in positive ion mode. Sodium iodide was used for mass calibration for a calibration range ofm/z 100–2500. Salt buffers from the protein samples were cleaned using manual syringe protein traps from Michrom BioResources (Auburn, CA). Proteins were prepared in a solution containing 50% acetonitrile, 50% water, 0.1% formic acid at a concentration of 50 pmol/μl and infused into the electrospray source at a rate of 5–10 μl min−1. Optimal ESI conditions were: capillary voltage of 3000 V, source temperature of 110 °C, and a cone voltage of 60 V. The ESI gas was nitrogen. Q1 was set to optimally pass ions from m/z 100–2000 and all ions transmitted into the pusher region of the time-of-flight analyzer were scanned over m/z 100–3000 with a 1-s integration time. Data were acquired in continuum mode until acceptable averaged data were obtained (10–15 min). ESI data were deconvoluted using MaxEnt I provided by Micromass. In-gel tryptic digests were performed on copper-stained protein bands (5, 10, and 20 μg of WT Tm IscU) separated on a 15% SDS-PAGE gel. Incised bands were washed with 50% methanol (HPLC-grade), 5% acetic acid (JT Baker UltrexII Ultrapure) and dried in HPLC-grade acetonitrile. Samples were then reduced with DTT (5 mg/ml in 100 mm ammonium bicarbonate) and alkylated with iodoacetamide (15 mg/ml in 100 mm ammonium bicarbonate). The protein band was dehydrated with acetonitrile, rehydrated with 100 mm ammonium bicarbonate, and dehydrated again with acetonitrile. Promega modified trypsin (20 ng/μl in 100 mm ammonium bicarbonate, total addition = 50 μl) was added to each gel piece and rehydrated on ice for 10 min. Samples were centrifuged and excess trypsin solution removed. Ammonium bicarbonate (50 mm) was added to 20 μl, vortexed and centrifuged briefly, and incubated at room temperature overnight. A solution of 50% acetonitrile, 5% formic acid (EM Science ACS 88%) was added to 30 μl, vortexed for 10 min, and centrifuged. The supernatant was isolated, and an additional 30 μl of 50% acetonitrile, 5% formic acid was added, vortexed for 10 min, and centrifuged. The supernatant was analyzed by matrix-assisted laser desorption/ionization time-of-flight performed on a Reflex III (Bruker, Bremen, Germany) mass spectrometer operated in linear, positive ion mode with a N2 laser. Laser power was used at the threshold level required to generate signal. Accelerating voltage was set to 28 kV. The instrument was calibrated with protein standards bracketing the molecular weights of the protein samples (typically mixtures of bradykinin fragment 1–5 and ACTH fragment 18–39 as appropriate). Salt buffers from the protein samples were cleaned using ZipTips (Millipore, Bedford, MA) according to manufacturer directions. α-Cyano-4-hydroxycinnamic acid was used as the matrix and prepared as a saturated solution in 50% acetonitrile, 0.1% trifluoroacetic acid (in water). Allotments of 1 μl of matrix and 1 μl of sample were thoroughly mixed together; 0.5 μl of this was spotted on the target plate and allowed to dry. Protein identification based on peptide masses was performed via ProFound (proteometrics.com). Protein (0.5 mm) in 50 mm sodium phosphate, pH 7.4, 50 mm NaCl, 50 mm DTT was repeatedly degassed and argon-purged for 1 h. Fresh FeCl3 and Na2S were then slowly added to 1 mm. The mixture was allowed to incubate anaerobically at room temperature for 0.5 h. Insoluble material was removed by centrifugation at 15,000 rpm, 4 °C for 5 min. The supernatant was desalted by a G-25 column equilibrated with 50 mm Tris-HCl, pH 7.4, or 50 mm sodium phosphate, pH 7.4, and the colored protein fraction was collected. His-tagged holoprotein was concentrated and stored at –80 °C until further use. Non-His-tagged holoprotein was then loaded onto a DE-52 column equilibrated with 50 mm Tris-HCl, pH 7.4, and washed with five column volumes of the same buffer. The holoprotein was eluted with 50 mm Tris-HCl, pH 7.4, 100 mm NaCl. FPLC gel filtration using a Superose-12 column (HR 16/50) at a flow rate of 0.5 ml/min was used for aggregation state determination. The running buffer was 50 mm HEPES, pH 7.4, 50 mm NaCl. A Gel Filtration Calibration Kit (Amersham Biosciences) was used to calibrate the column. The standards were RNase A (13,700 Da), chymotrypsinogen A (25,000 Da), ovalbumin (43,000 Da), and albumin (67,000 Da). Blue dextrin was used to determine the dead volume. Molecular sizes were determined by plotting log Mr of standardsversus Kav whereKav = (Ve −V0)/(Vt −V0), Ve = elution volume, V0 = dead volume, Vt = total column volume. UV-visible spectra were recorded on a Hewlett-Packard 8425A diode array spectrophotometer using the On-Line Instrument Systems (OLIS) 4300S operating system software. A 1.0-cm path-length cuvette was used for all measurements. All solutions used for extinction coefficient determination were prepared in Barnstead purified deionized water. Apo D40A Tm IscU was initially dialyzed extensively against a volatile buffer (100 mmammonium bicarbonate, pH 7.0) and then against unbuffered water. Finally, the protein was passed through a G-25 column equilibrated with water. After the absorption spectrum was collected, the sample was lyophilized and the mass of the protein sample determined. Using Beer's law and the molecular mass determined by ESI, the extinction coefficient of apo D40A Tm IscU was calculated. The concentration of protein used for holo D40A Tm IscU extinction coefficient calculation was determined by incubating holo D40A Tm IscU in 50 mm Tris-HCl, pH 7.4, 50 mm NaCl, 1 mm EDTA at 60 °C for 0.5 h followed by desalting via a G-25 column. Total protein concentration was then determined from the 278-nm absorption of apo D40ATm IscU using the previously determined extinction coefficient. Holoprotein (30 μm) in 100 mmTris-HCl, pH 7.4, 50 mm NaCl was repeatedly degassed and argon-purged in a stoppered cuvette while kept on ice. Absorption data at 412 nm was acquired on a Hewlett-Packard diode array spectrometer (HP 8453) with HP8453 Win system software. Temperature control was achieved with a Peltier temperature controller (HP 8909A). Absorption data at temperatures ranging from 20 °C to 86 °C was collected at 2 °C increments with a 0.5-min equilibration period at each temperature prior to measurement. Mössbauer spectra of 57Fe D40A Tm IscUHis was recorded on a constant acceleration spectrometer, model MS-1200D from Ranger Scientific, using a Janis SuperVaritemp cryostat (model 8DT), a Lakeshore temperature controller (model 340), and a 57Co source from Isotope Products Laboratory. The57Fe-reconstituted protein was prepared as described above (see Tm IscU cluster reconstitution) except that57Fe3+ was used instead of regular FeCl3. EPR signals were recorded with an X-band Bruker ESP 300 spectrometer equipped with an Oxford liquid helium cryostat at 15 K. Holo D40A Tm IscUHis concentrations were 0.9 mm for untreated samples, and 0.4 mm for ascorbate reduced samples. Holo D40A Tm IscUHis was reduced with 0.2 mm and 2 mm ascorbate and immediately frozen. 15N-Labeled apo D40A Tm IscU was expressed in minimal medium supplemented with15NH4Cl (22Agarwal A. Tan J. Eren M. Tevlev A. Lui S.M. Cowan J.A. Biochem. Biophys. Res. Commun. 1994; 197: 1357-1362Crossref Scopus (30) Google Scholar). 15N-HSQC spectra were recorded on a Bruker 600 Avance DMX spectrometer operating at 600.13 MHz. The pulse sequence was as described previously (23Bodenhausen G. Ruben D.J. Chem. Phys. Lett. 1980; 69: 185Crossref Scopus (2430) Google Scholar). A total of four scans were collected. Iron content of holo D40A TmIscU was determined by atomic absorption using a PerkinElmer Zeeman 5000 graphite furnace atomic absorption spectrometer. An iron standard solution (GFS Chemicals, Inc.) was used to construct a standard curve with a r2 > 0.999. Sample loading was automated (AS40), and all data were run in duplicate and averaged. Absorption was measured at 305.9 nm and integrated for 7 s. All solutions were in Barnstead purified deionized water and 2% nitric acid. Background iron concentrations of solutions were measured and found to be negligible. Circular dichroism spectra were measured on an Aviv model 202 circular dichroism spectrometer. Far-UV CD spectra were acquired with a 0.1-mm path-length cuvette. Experimental conditions were essentially as recommended by Johnson (24Johnson W.C.J. Proteins Struct. Funct. Genet. 1990; 7: 205-214Crossref PubMed Scopus (894) Google Scholar). Protein concentrations were 0.08 mm WT and D40A Tm IscU (based on monomeric protein) and 0.15 mmHs Fd in 10 mm sodium phosphate, pH 7.0, 10 mm NaCl, except for holo D40A Tm IscU (10 mm Tris-HCl, pH 7.0, 50 mm NaCl). Spectra acquired at 20 °C were determined per 0.2 nm in triplicate and averaged. At elevated temperatures spectra were only recorded once. Secondary structure quantitation was determined via the self-consistent method (25Sreerama N. Woody R.W. J. Mol. Biol. 1994; 242: 497-501PubMed Google Scholar) with the Dicroprot V2.5 version 5.0 package (26Deleage G. Geourjon C. Comp. Appl. Biosci. 1993; 9: 197-199PubMed Google Scholar) obtained from www.ibcp.fr. Near-UV-visible CD spectra were recorded in a 3-mm path-length cuvette/1 nm. Protein concentrations were 40 μm in 50 mm Tris-HCl, pH 7.0. Buffer spectra were always subtracted. Reactions were initiated by the addition of 0.1 mm holo D40A Tm IscUHis in 50 mmsodium phosphate, pH 7.4, to a solution containing 0.1 mmhuman apoferredoxin and 5 mm DTT in the same buffer. Reactions were incubated on ice, stopped by the addition of loading buffer, and immediately frozen at –80 °C. Reaction products were separated on a 7% native-PAGE and visualized by Coomassie Blue staining. Tm IscU expression was high for all constructs in BL21CodonPlus(DE3)-RIL, greater than that obtained from BL21(DE3), with typical yields of greater than 700 mg of pure protein from a 10-liter fermentation. BL21CodonPlus(DE3)-RIL provides tRNAs for the rarely used E. coli codons of Arg, Ile, and Leu. These codons are more common inT. maritima and are found within the iscUgene. Surprisingly, expressed WT Tm IscU and His-tagged WT Tm IscU (Tm IscUHis) were observed to run as two closely spaced bands during SDS-PAGE. These bands were found in a 1:1 ratio and persisted from the time of lysis through all purification steps (Fig. 1) and give rise to similar mass spectrometric patterns (see below). Phenylmethylsulfonyl fluoride had no effect on the appearance or ratio of the two bands, and so they do not seem to be a result of endoproteinase activity. D40A Tm IscU and D40A TmIscUHis only expressed as one band migrating at the same position as the lighter molecular mass band of the corresponding WT protein. Apo WT and D40A Tm IscU migrated as a monomer on an analytical gel filtration column, whereas holo WT and D40ATm IscU eluted as a dimer with apparent masses of 18 and 35 kDa, respectively (Fig. 2). No high molecular mass aggregates were observed to elute. ESI spectra of each of the two bands identified by SDS-PAGE for WT Tm IscU showed the same two major peaks corresponding to full-length protein and to protein lacking the amino-terminal Met in approximately a 1:1 ratio (16,070 and 15,940 Da, respectively), whereas D40A Tm IscU showed complete loss of the amino-terminal Met with a mass of 15,893 Da. These values are in close agreement with their predicted molecular masses of 16,071, 15,940, and 15,894 Da, respectively. In-gel tryptic digests of the two bands of WT Tm IscU yielded essentially identical matrix-assisted laser desorption/ionization-time of flight spectra for all three protein concentrations tested, i.e. peptide fragments with identical masses were observed for both SDS-PAGE bands without the appearance or disappearance of any signal. The search engine ProFound identified both bands as consisting of TmIscU, further confirming that neither of the bands was caused by an impurity. The bacterial pellets of induced cells were dark brown in color for all constructs. Following centrifugation to remove solids, His-tagged WT and D40A Tm IscU lysates yielded a red band that was bound by a Ni-NTA column. The non-His-tagged constructs had reddish-brown lysates, but no color survived the 85 °C purification step. These results suggest proper Fe-S cluster assembly in vivo in E. coli, and that the His tag does not interfere with Fe-S cluster coordination. Although a fraction of Tm IscU expressed as holoprotein, the overall holo concentration appeared to be quite low as judged by UV-visible spectroscopy. Therefore, attempts were made to reconstituteTm IscU. Simple anaerobic incubation of either apo D40ATm IscU or D40A Tm IscUHis with DTT and a 2-fold molar excess of iron and sulfide without the presence of denaturant resulted in a deep red protein with UV-visible spectra similar to [2Fe-2S]-containing proteins (Fig. 3). Increasing the iron and sulfide concentration only resulted in the appearance of adventitiously bound iron, as judged by Mössbauer analysis (data not shown). Holo D40A Tm IscU was further purified by anion exchange chromatography. Apo D40A Tm IscU did not bind to DE-52, whereas holo D40A Tm IscU was o" @default.
• W2094139743 created "2016-06-24" @default.
• W2094139743 creator A5011309627 @default.
• W2094139743 creator A5024364711 @default.
• W2094139743 creator A5044505147 @default.
• W2094139743 creator A5053218662 @default.
• W2094139743 date "2002-06-01" @default.
• W2094139743 modified "2023-10-03" @default.
• W2094139743 title "Iron-Sulfur Cluster Biosynthesis" @default.
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