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• W1964152102 abstract "Core-binding factors (CBF) are heteromeric transcription factors essential for several developmental processes, including hematopoiesis. CBFs contain a DNA-binding CBFα subunit and a non-DNA binding CBFβ subunit that increases the affinity of CBFα for DNA. We have developed a procedure for overexpressing and purifying full-length CBFβ as well as a truncated form containing the N-terminal 141 amino acids using a novel glutaredoxin fusion expression system. Substantial quantities of the CBFβ proteins can be produced in this manner allowing for their biophysical characterization. We show that the full-length and truncated forms of CBFβ bind to a CBFα·DNA complex with very similar affinities. Sedimentation equilibrium measurements show these proteins to be monomeric. Circular dichroism spectroscopy demonstrates that CBFβ is a mixed α/β protein and NMR spectroscopy shows that the truncated and full-length proteins are structurally similar and suitable for structure determination by NMR spectroscopy. Core-binding factors (CBF) are heteromeric transcription factors essential for several developmental processes, including hematopoiesis. CBFs contain a DNA-binding CBFα subunit and a non-DNA binding CBFβ subunit that increases the affinity of CBFα for DNA. We have developed a procedure for overexpressing and purifying full-length CBFβ as well as a truncated form containing the N-terminal 141 amino acids using a novel glutaredoxin fusion expression system. Substantial quantities of the CBFβ proteins can be produced in this manner allowing for their biophysical characterization. We show that the full-length and truncated forms of CBFβ bind to a CBFα·DNA complex with very similar affinities. Sedimentation equilibrium measurements show these proteins to be monomeric. Circular dichroism spectroscopy demonstrates that CBFβ is a mixed α/β protein and NMR spectroscopy shows that the truncated and full-length proteins are structurally similar and suitable for structure determination by NMR spectroscopy. The core-binding factor β subunit (CBFβ) 1The abbreviations used are: CBFβ, core-binding factor β subunit; CBFβ(141), the 143-amino acid protein containing residues Gly-Ser followed by the N-terminal 141 amino acids of murine CBFβ; CBFβ(187), the 189-amino acid protein containing residues Gly-Ser followed by amino acids encoding the 187-amino acid isoform of CBFβ; DTT, dithiothreitol; Grx, E. coli glutaredoxin-1; HSQC, heteronuclear single quantum coherence; NH, protein backbone amide NH; PAGE, polyacrylamide gel electrophoresis; MALDI, matrix-assisted laser desorption ionization. is the non-DNA binding subunit of the heterodimeric transcription factor complex called core binding factor (CBF) (1Ogawa E. Inuzuka M. Maruyama M. Satake M. Naito-Fujimoto M. Ito Y. Shigesada K. Virology. 1993; 194: 314-331Crossref PubMed Scopus (442) Google Scholar, 2Wang S. Wang Q. Crute B.E. Melnikova I.N. Keller S.R. Speck N.A. Mol. Cell. Biol. 1993; 13: 3324-3339Crossref PubMed Scopus (397) Google Scholar). CBF binds to target DNA sites with the consensus sequence PyGPyGGT (3Kamachi Y. Ogawa E. Asano M. Ishida S. Murakami Y. Satake M. Ito Y. Shigesada K. J. Virol. 1990; 64: 4808-4819Crossref PubMed Google Scholar, 4Melnikova I.N. Crute B.E. Wang S. Speck N.A. J. Virol. 1993; 67: 2408-2411Crossref PubMed Google Scholar, 5Thornell A. Hallberg B. Grundstrom T. J. Virol. 1991; 65: 42-50Crossref PubMed Google Scholar, 6Meyers S. Downing J.R. Hiebert S.W. Mol. Cell. Biol. 1993; 13: 6336-6345Crossref PubMed Scopus (427) Google Scholar). CBF subunits are encoded by four genes in mammals. CBFA1, CBFA2(AML1), and CBFA3 encode DNA-binding CBFα subunits, and CBFB encodes the common CBFβ subunit (1Ogawa E. Inuzuka M. Maruyama M. Satake M. Naito-Fujimoto M. Ito Y. Shigesada K. Virology. 1993; 194: 314-331Crossref PubMed Scopus (442) Google Scholar, 2Wang S. Wang Q. Crute B.E. Melnikova I.N. Keller S.R. Speck N.A. Mol. Cell. Biol. 1993; 13: 3324-3339Crossref PubMed Scopus (397) Google Scholar,7Bae S.C. Takahashi E. Zhang Y.W. Ogawa E. Shigesada K. Namba Y. Satake M. Ito Y. Gene (Amst.). 1995; 159: 245-248Crossref PubMed Scopus (152) Google Scholar, 8Bae S.C. Yamaguchi-Iwai Y. Ogawa E. Maruyama M. Inuzuka M. Kagoshima H. Shigesada K. Satake M. Ito Y. Oncogene. 1993; 8: 809-814PubMed Google Scholar, 9Levanon D. Negreanu V. Bernstein Y. Bar-Am I. Avivi L. Groner Y. Genomics. 1994; 23: 425-432Crossref PubMed Scopus (381) Google Scholar, 10Liu P. Tarle S.A. Claxton D.F. Marlton P. Freedman M. Siciliano M.J. Collins F.S. Science. 1993; 261: 1041-1044Crossref PubMed Scopus (651) Google Scholar, 11Miyoshi H. Shimizu K. Kozu T. Maseki N Kaneko Y. Ohki M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10431-10434Crossref PubMed Scopus (800) Google Scholar, 12Ogawa E. Maruyama M. Kagoshima H. Inuzuka M. Lu J. Satake M. Shigesada K. Ito Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6859-6863Crossref PubMed Scopus (562) Google Scholar). The CBFα subunit can bind DNA in the absence of CBFβin vitro but with 5–10-fold lower affinity than the CBFα/β complex (1Ogawa E. Inuzuka M. Maruyama M. Satake M. Naito-Fujimoto M. Ito Y. Shigesada K. Virology. 1993; 194: 314-331Crossref PubMed Scopus (442) Google Scholar, 2Wang S. Wang Q. Crute B.E. Melnikova I.N. Keller S.R. Speck N.A. Mol. Cell. Biol. 1993; 13: 3324-3339Crossref PubMed Scopus (397) Google Scholar). CBFβ modulates the affinity of the CBFα subunit for DNA without establishing additional contacts on the DNA or changing the magnitude of DNA bending (2Wang S. Wang Q. Crute B.E. Melnikova I.N. Keller S.R. Speck N.A. Mol. Cell. Biol. 1993; 13: 3324-3339Crossref PubMed Scopus (397) Google Scholar, 13Golling G. Li L.H. Pepling M. Stebbins M. Gergen J.P. Mol. Cell. Biol. 1996; 16: 932-942Crossref PubMed Google Scholar). The amino acid sequence of CBFβ yields few clues to its tertiary structure or the mechanism by which it modulates the affinity of the CBFα subunit for DNA. The heterodimerization domain in CBFβ has been localized to its N-terminal 135 amino acids, which corresponds to a region of significant homology between CBFβ and its twoDrosophila homologs, Brother (Bro) and Big Brother (Bgb) (13Golling G. Li L.H. Pepling M. Stebbins M. Gergen J.P. Mol. Cell. Biol. 1996; 16: 932-942Crossref PubMed Google Scholar, 14Kagoshima H. Akamatsu Y. Ito Y. Shigesada K. J. Biol. Chem. 1996; 271: 33074-33082Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). A truncated CBFβ protein containing amino acids 1–141 (CBFβ(141)), which includes the region of homology to Bro and Bgb, stably associates with the CBFα subunit in vitro (1Ogawa E. Inuzuka M. Maruyama M. Satake M. Naito-Fujimoto M. Ito Y. Shigesada K. Virology. 1993; 194: 314-331Crossref PubMed Scopus (442) Google Scholar, 14Kagoshima H. Akamatsu Y. Ito Y. Shigesada K. J. Biol. Chem. 1996; 271: 33074-33082Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar,15Wang Q. Stacy T. Miller J.D. Lewis A.F. Huang X. Bushweller J.H. Boris J.-C. Alt F.W. Ryan G. Liu P.P. Wynshaw-Boris A. Binder M. Marı́n-Padilla M. Sharpe A. Speck N.A. Cell. 1996; 87: 697-708Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar). Further truncation of the C terminus to amino acid 133 results in a protein that weakly associates with CBFα, and C-terminal truncation to amino acid 117 disrupts stable heterodimerization with CBFα (1Ogawa E. Inuzuka M. Maruyama M. Satake M. Naito-Fujimoto M. Ito Y. Shigesada K. Virology. 1993; 194: 314-331Crossref PubMed Scopus (442) Google Scholar,15Wang Q. Stacy T. Miller J.D. Lewis A.F. Huang X. Bushweller J.H. Boris J.-C. Alt F.W. Ryan G. Liu P.P. Wynshaw-Boris A. Binder M. Marı́n-Padilla M. Sharpe A. Speck N.A. Cell. 1996; 87: 697-708Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar). The CBFβ subunit is essential for the in vivo function of at least one of the CBFα subunits that were encoded by theCBFA2 gene (also known as the acute myeloid leukemia 1 orAML1 gene). Homozygous disruption of either theCbfa2 or the Cbfb genes in mice results in identical phenotypes: midgestation embryonic lethality accompanied by extensive hemorrhaging and a profound block at the fetal liver stage of hematopoiesis (15Wang Q. Stacy T. Miller J.D. Lewis A.F. Huang X. Bushweller J.H. Boris J.-C. Alt F.W. Ryan G. Liu P.P. Wynshaw-Boris A. Binder M. Marı́n-Padilla M. Sharpe A. Speck N.A. Cell. 1996; 87: 697-708Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar, 16Niki M. Okada H. Takano H. Kuno J. Tani K. Hibino H. Asano S. Ito Y. Satake M. Noda T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5697-5702Crossref PubMed Scopus (164) Google Scholar, 17Okuda T. van Deursen J. Hiebert S.W. Grosveld G. Downing J.R. Cell. 1996; 84: 321-330Abstract Full Text Full Text PDF PubMed Scopus (1606) Google Scholar, 18Sasaki K. Yagi H. Bronson R.T. Tominaga K. Matsunashi T. Deguchi K. Tani Y. Kishimoto T. Komori T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12359-12363Crossref PubMed Scopus (328) Google Scholar, 19Wang Q. Stacy T. Binder M. Marı́n-Padilla M. Sharpe A.H. Speck N.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3444-3449Crossref PubMed Scopus (1029) Google Scholar). In humans, chromosomal rearrangements that disrupt the CBFA2 and CBFB genes are associated with a variety of leukemias, including de novo acute myeloid leukemias t(8;21)(q22;q22), inv(16)(p13;q22), t(16;16), and del(16)(q22), acute lymphocytic leukemias t(12;21)(p13;q22), and therapy-related leukemias and myelodysplasias t(3;21)(q26;q22) (10Liu P. Tarle S.A. Claxton D.F. Marlton P. Freedman M. Siciliano M.J. Collins F.S. Science. 1993; 261: 1041-1044Crossref PubMed Scopus (651) Google Scholar, 11Miyoshi H. Shimizu K. Kozu T. Maseki N Kaneko Y. Ohki M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10431-10434Crossref PubMed Scopus (800) Google Scholar,20Golub T.R. Barker G.F. Bohlander S.K. Hiebert S. Ward D.C. Bray-Ward P. Morgan E. Raimondi S.C. Rowley J.D. Gilliland D.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4917-4921Crossref PubMed Scopus (673) Google Scholar, 21Nucifora G. Begy C.R. Erickson P. Drabkin H.A. Rowley J.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7784-7788Crossref PubMed Scopus (179) Google Scholar, 22Romana S.P. Mauchauffe M. Le Coniat M. Chumakow I. Le Paslier D. Berger R. Bernard O.A. Blood. 1995; 85: 3662-3670Crossref PubMed Google Scholar). All of these translocations result in the synthesis of chimeric proteins, two of which have been directly demonstrated to block CBF function in a transdominant manner (23Yergeau D.A. Hetherington C.J. Wang Q. Zhang P. Sharpe A.H. Binder M. Marin-Padilla M. Speck N.A. Zhang D.-E. Nat. Genet. 1997; 15: 303-306Crossref PubMed Scopus (310) Google Scholar, 24Castilla L.H. Wijmenga C. Wang Q. Stacy T. Speck N.A. Eckhaus M. Marin-Padilla M. Collins F.S. Wynshaw-Boris A. Liu P.P. Cell. 1997; 87: 687-696Abstract Full Text Full Text PDF Scopus (251) Google Scholar). The primary structures of CBFβ and its Drosophila homologs are not similar to those of any other proteins, and the mechanism by which CBFβ stabilizes the CBFα·DNA complex is unusual. The CBFβ subunit is an essential component of the CBF complex and is mutated in a significant percentage of human leukemias, making it both an interesting and important target for biophysical and structural analyses. In this study, we describe a novel system for expressing the CBFβ subunit in bacteria and a purification protocol with which we can obtain large amounts of homogeneous CBFβ protein. We confirm that a truncated form of CBFβ, CBFβ(141), demonstrated previously to contain an intact heterodimerization domain (1Ogawa E. Inuzuka M. Maruyama M. Satake M. Naito-Fujimoto M. Ito Y. Shigesada K. Virology. 1993; 194: 314-331Crossref PubMed Scopus (442) Google Scholar), binds to CBFα with an affinity very similar to that of the full-length CBFβ(187) protein. The isolated heterodimerization domain in CBFβ(141) also assumes a folded structure indistinguishable from that which it assumes in the context of the full-length CBFβ(187) protein. We also demonstrate that both the full-length CBFβ(187) and truncated CBFβ(141) proteins are monomeric and contain a mixture of α-helical and β-strand secondary structural elements. The pGRXCBFβ141 plasmid encoding the glutaredoxin-CBFβ(141) fusion protein was constructed in the following manner. DNA sequence corresponding to Escherichia coli glutaredoxin-1 was polymerase chain reaction amplified from the plasmid pMG524-GRX (25Bjornberg O. Holmgren A. Protein Expression Purif. 1991; 2: 287-295Crossref PubMed Scopus (26) Google Scholar) using the following primers: 5′-CGGAATTCGGTTAAACCTACTTTCAGCG-3′ (S-GRX) and 5′-CGGGATCCCTTGTCATCGTCATCGGCGTCCAGATTTTCTTTCACC-3′ (AS-GRX). The sense primer (S-GRX) contains the recognition site for EcoRI at its 5′ end. The AS-GRX primer contains a restriction site forBamHI, followed by sequences encoding a cleavage site for the protease enterokinase (DDDDK). DNA encoding CBFβ(141) was amplified from the plasmid CBFβ(p21.5) (2Wang S. Wang Q. Crute B.E. Melnikova I.N. Keller S.R. Speck N.A. Mol. Cell. Biol. 1993; 13: 3324-3339Crossref PubMed Scopus (397) Google Scholar) using the primers 5′-AGACGGATCCATGCCGCCGTCGTCCCGGAC-3′ (S-CBFβ141) and 5′-GGCCCAAGCTTTCACTGTTGTGCTAATGCATCTTCC-3′ (AS-CBFβ141). A restriction site for BamHI was included at the 5′ end of the sense primer (S-CBFβ141), and a HindIII site followed by a translational stop codon was added to the antisense primer (AS-CBFβ141). The polymerase chain reaction amplified fragments were digested with the appropriate restriction enzymes and subcloned between the EcoRI and HindIII sites of pMG524. The resultant plasmid was transformed into the bacterial strain N99cI for the purpose of DNA characterization and sequencing and into strain N4830 or AR58 for protein expression. The pGRXCBFβ187 plasmid encoding the glutaredoxin protein fused to CBFβ(1–187) was generated by replacing theBamHI-HindIII fragment in pGRXCBFβ141 that encodes CBFβ(141) with a BamHI-HindIII fragment containing the open reading frame of CBFβ(1–187), which was excised from a previously described plasmid containing CBFβ(1–187) subcloned into pBluescript SK+ (2Wang S. Wang Q. Crute B.E. Melnikova I.N. Keller S.R. Speck N.A. Mol. Cell. Biol. 1993; 13: 3324-3339Crossref PubMed Scopus (397) Google Scholar). A cleavage site for Factor Xa was subsequently introduced into this plasmid. Complementary oligonucleotides encoding a Factor Xa cleavage site flanked byBamHI sites (5′-GATCCATCGAAGGTCGTG-3′ and 5′-GATCCACGACCTTCGATG-3′) were annealed, phosphorylated, and subcloned into the BamHI site, between the enterokinase cleavage site and the open reading frame of CBFβ(187). The inserts in all plasmids were confirmed by sequencing. These plasmids are available upon request to the corresponding authors. E. coli AR58 were transformed with the pGRXCBFβ141 plasmid, and a single colony was used to inoculate 10 ml of terrific broth (Sigma) containing 100 μg/ml carbenicillin. After overnight shaking at 200 rpm at a temperature of 29 °C, the culture was used to inoculate 1 liter of terrific broth (100 μg/ml carbenicillin). This culture was grown to an A 600 of 1.5 and then induced by raising the temperature to 40 °C and maintaining at this temperature for 4 h. Cells were collected by centrifugation, resuspended in an equal weight of 10% sucrose, 50 mm Tris (pH 7.5), and frozen by dripping into liquid nitrogen. The frozen cells were stored at −70 °C. For expression of full-length CBFβ, pGRXCBFβ187 was transformed into E. coli AR58, and cells were grown in the identical manner as for CBFβ(141). For expression of 15N-labeled CBFβ(141) or CBFβ(187), E. coli AR58 transformed with the appropriate plasmid was grown in minimal medium containing15(NH4)2SO4 (Isotec, Inc.) as the sole nitrogen source. All operations were carried out at 4 °C. Pefabloc (1 mm, Boehringer Mannheim), EDTA (1 mm), and lysozyme (0.4 mg/ml) were added to the thawed bacterial cell pellet from a 1-liter culture. The solution was stirred gently for 30 min, and the cells were then further lysed by four passages through a French press at 18,000 p.s.i. The lysate was clarified by centrifugation at 11,000 × g for 10 min. The resulting supernatant was loaded onto a 2.5 × 25-cm column of DEAE-Sephacel (Pharmacia Biotech Inc.) equilibrated in 25 mm Tris-Cl (pH 7.5), 1 mm EDTA, 1 mm DTT at 1 ml/min and eluted from the column with a 1-liter linear gradient of 0–500 mm NaCl in the same buffer. Fractions containing the fusion protein were identified by hydroxyethyl disulfide assay, which detects the enzymatic activity of the glutaredoxin system as described by Holmgren (26Holmgren A. J. Biol. Chem. 1979; 254: 3664-3671Abstract Full Text PDF PubMed Google Scholar). The active fractions were pooled and concentrated by ultrafiltration on a YM3 membrane (Amicon, Inc.) to 15 ml. The DEAE pool was loaded onto a 2.5 × 113-cm column of Sephacryl S-100 (Pharmacia) in 25 mm Tris-Cl (pH 7.5), 1 mm EDTA, 75 mm NaCl and eluted with the same buffer at 0.6 ml/min. The active fractions were again identified by 2-hydroxyethyl disulfide assay, pooled, and concentrated by ultrafiltration to 15 ml. The resulting homogeneous fusion protein was cleaved by treatment with 90 units of enterokinase (Biozyme, San Diego)/A 280unit of fusion protein with addition of DTT to 0.5 mm and incubation at 30 °C for 20 h. The cleavage reaction was halted by addition of 0.5 mm Pefabloc. The resulting cleaved protein was exchanged into a lower ionic strength buffer (25 mm NaCl), loaded onto a 2.5 × 10-cm column of Q-Sepharose (Pharmacia) in 25 mm Tris-Cl (pH 7.5), 1 mm EDTA, 25 mm NaCl, 1 mm DTT, and eluted with a 500-ml linear gradient of 25–500 mm NaCl in the same buffer at 0.8 ml/min. The CBFβ fractions were identified by SDS-PAGE, pooled, and concentrated to 10 ml by ultrafiltration. From 1 liter of culture, a yield of 20 mg is typically obtained. For the15N-labeled protein used for NMR studies, the Q-Sepharose pool was loaded onto a 2.5 × 36-cm column of Sephacryl S-100 in 25 mm potassium phosphate (pH 6.5), 0.1 mmEDTA, 1 mm DTT, 0.1% NaN3 and eluted at 0.8 ml/min. The protein was concentrated by ultrafiltration. All operations were carried out at 4 °C. The Grx-CBFβ(187) fusion protein was purified in the same manner as described above for the Grx-CBFβ(141) fusion protein. Following fractionation on DEAE-Sephacel and Sephacryl S-100, the resulting homogeneous Grx-CBFβ187 fusion protein was cleaved by treatment with 3.5 units of Factor Xa (Pharmacia)/A 280 unit of fusion protein in a buffer containing 25 mm Tris-Cl (pH 7.5), 1 mmEDTA, 75 mm NaCl, 1 mm CaCl2, and no DTT. Secondary cleavage at an undesired Factor Xa-sensitive site was minimized by carrying out the reaction for 6 h at 22 °C. The cleavage reaction was halted by addition of 4 mm Pefabloc with a subsequent 40-min incubation at 37 °C. The cleaved protein was exchanged into a lower ionic strength buffer (25 mmNaCl) by ultrafiltration, loaded onto a 2.5 × 10-cm column of Q-Sepharose in 25 mm Tris-Cl (pH 7.5), 1 mmEDTA, 25 mm NaCl, 1 mm DTT, and eluted with a 500-ml linear gradient from 25 to 500 mm NaCl in the same buffer at 0.8 ml/min. The CBFβ(187) fractions were identified by SDS-PAGE, pooled, and concentrated to 4 ml by ultrafiltration. The concentrated protein was loaded onto a 2.5 × 113-cm column of Sephacryl S-100 (Pharmacia) in 25 mm Tris (pH 7.5), 1 mm EDTA, 1 mm DTT, 75 mm NaCl and eluted with the same buffer to separate the full-length protein from the product of the secondary cleavage reaction. SDS-polyacrylamide gel electrophoresis was carried out using 15% Ready Gels from Bio-Rad with Coomassie staining as described by the manufacturer. Isoelectric focusing gel electrophoresis was carried out on a Pharmacia Phast System using IEF 3–9 gels, and Coomassie staining was performed as described by the manufacturer. The molar extinction coefficients for the CBFβ(141) and CBFβ(187) proteins were calculated on the basis of the tryptophan, tyrosine, and cystine content of the proteins as described by Pace et al. (27Pace N.C. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3450) Google Scholar). A value of 18,500 m−1 cm−1 was calculated for both proteins. MALDI mass spectrometry was carried out by Dr. Gary Siuzdak at the mass spectrometry laboratory at the Beckman Center for Chemical Sciences, Scripps Research Institute. Samples were dialyzed into 20 mm ammonium acetate (pH 7), 0.5 mm DTT prior to analysis. To confirm the site of cleavage by enterokinase, N-terminal sequencing of the first five residues was carried out at the Biotechnology Resource Laboratory located at Yale University. A sequence of Xaa-Xaa-Met-Pro-Arg was obtained showing that cleavage had occurred at the correct site; however, the sequence of the first two amino acids was not determined with certainty. The affinities of CBFβ(141) and CBFβ(187) for a CBFα·DNA complex were measured by electrophoretic mobility shift assay. The source of CBFα2 was an 41–214-amino acid fragment from the murine CBFα2 protein encompassing the DNA-binding Runt domain, expressed in bacteria and purified as described previously (28Crute B.E. Lewis A.F. Wu Z. Bushweller J.H. Speck N.A. J. Biol. Chem. 1996; 271: 26251-26260Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Basically, binding conditions were chosen such that the concentration of DNA was >10-fold above theK d of the CBFα2 Runt domain for the DNA site (∼1 × 10−8m DNA), and the concentration of active CBFα2 Runt domain was >10-fold below theK d of CBFβ for the CBFα2·DNA complex [5 × 10−10m CBFα2]. A fixed amount of DNA and the Runt domain, along with various amounts of CBFβ proteins, were used in binding reactions, and the complexes were resolved by electrophoretic mobility shift assay. Assay conditions and methods for quantitation were the same as described previously (15Wang Q. Stacy T. Miller J.D. Lewis A.F. Huang X. Bushweller J.H. Boris J.-C. Alt F.W. Ryan G. Liu P.P. Wynshaw-Boris A. Binder M. Marı́n-Padilla M. Sharpe A. Speck N.A. Cell. 1996; 87: 697-708Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar). The data are plotted as the percentages of CBFα2·CBFβ·DNA complexversus the concentration of CBFβ, and theK d is defined as the concentration of CBFβ at 50% saturation (Kaleidagraph, Synergy Software). 100% ternary complex was defined as the point of saturation, and 0% ternary complex is taken from the background at the position in the absence of added CBFβ. High speed (29Yphantis D.A. Biochemistry. 1964; 3: 297-317Crossref PubMed Scopus (2019) Google Scholar) sedimentation equilibrium experiments were conducted at 20 °C in a Beckman XL-l ultracentrifuge at rotor speeds of 15,000, 20,000, 25,000, and 30,000 rpm (CBFβ(141)) or 10,000, 15,000, 20,000, and 25,000 rpm (CBFβ(187)) using absorbance detection. For each sample, three cell loading concentrations were examined, from 0.25 to ∼1 mg/ml in 25 mm Tris-Cl (pH 7.5), 150 mm KCl, 1 mm DTT. Data were fit using NONLIN (30Johnson M.L. Correia J.J. Yphantis D.A. Halvorson H.R. Biophys. J. 1981; 36: 575-588Abstract Full Text PDF PubMed Scopus (778) Google Scholar). Partial specific volumes and solvent density (1.00960 g/ml) were calculated as described (31Laue T.M. Short Column Sedimentation Equilibrium Analysis for Rapid Characterization of Macromolecules in Solution, Technical Information DS-835. Beckman Instruments Inc., Palo Alto, CA1992Google Scholar). For CBFβ(141), the calculated partial specific volume (0.7225 ml/g) was adjusted for the peptide charge (−3 at pH 7.5) by decreasing the molar volume 25 ml/mol charge to 0.7185 ml/g. An additional uncertainty of 0.004 ml/g in the partial specific volume was included when calculating the molecular weights (32Starovasnik M.A. Blackwell T.K. Laue T.M. Weintraub H. Klevit R.E. Biochemistry. 1992; 31: 9891-9903Crossref PubMed Scopus (33) Google Scholar, 33Kharakoz D.P. Biophys. Chem. 1989; 34: 115-125Crossref PubMed Scopus (204) Google Scholar). For CBFβ(187), the calculated partial specific volume (0.7161 ml/g) also was reduced for its expected charge (−4 at pH 7.5) by 0.004 ml/g, and the same additional uncertainty was included in the molecular weight calculations. Circular dichroism spectra were collected at 20 °C on a Jasco 715 spectrometer calibrated using 10-camphorsulfonic acid. Mean amide Δε values were calculated using the known protein sequence. The protein solutions were dialyzed extensively against 25 mm potassium phosphate (pH 7.5), 0.1 mm EDTA, 0.5 mm DTT prior to CD measurements. Quartz cells of 0.05 mm were used for measurements in the far ultraviolet (176–260 nm). The data were corrected by subtraction of a spectrum of the buffer alone. A total of eight scans were recorded at 1 nm resolution from 265 to 175 nm for both protein and buffer at a rate of 10 nm/min with a 16-s response time. The resulting data for 178–260 nm were fit using the variable selection protocol of Johnson and co-worker (34Johnson W.C. Proteins Struct. Funct. Genet. 1990; 7: 205-214Crossref PubMed Scopus (894) Google Scholar, 35Manavalan P. Johnson W.C. Anal. Biochem. 1987; 167: 76-85Crossref PubMed Scopus (664) Google Scholar) using software provided by Dr. Johnson. Three proteins at a time were removed from the 33-protein data base, and the resulting 5456 combinations were examined for total percentage of secondary structure and root mean square error. Eleven combinations were finally selected, all of which had root mean square error values less than 0.20 (CBFβ(141)) and 0.25 (CBFβ(187)). All measurements were performed on a Varian UnityPlus 500 NMR spectrometer equipped with an actively shielded triple-resonance probe from Nalorac Corporation and pulsed field gradients. Measurements were carried out at 20 °C with solutions of the proteins in 25 mm potassium phosphate (pH 6.5), 0.1 mm EDTA, 0.1% sodium azide, 5 mmDTT. 15N-1H HSQC spectra were recorded using the gradient sensitivity-enhanced HSQC sequence (36Kay L.E. Keifer P. Saarinern T. J. Am. Chem. Soc. 1992; 114: 10663-10665Crossref Scopus (2432) Google Scholar). The number of complex points and acquisition times for these experiments were15N (F1), 128 points, 79.3 ms and 1H (F2), 1024 points, 157 ms. For measurement of 15N T 1 andT 2 values, the pulse sequences of Farrowet al. (37Farrow N.A. Muhandiram R. Singer A.U. Pascal S.M. Kay C.M. Gish G. Shoelson S.E. Pawson T. Forman-Kay J.D. Kay L.E. Biochemistry. 1994; 33: 5984-6003Crossref PubMed Scopus (2013) Google Scholar) optimized for minimal saturation of water were employed. A recycle delay of 1.0 s was used between acquisitions to ensure sufficient recovery of NH magnetization (38Sklenar V. Torchia D.A. Bax A. J. Magn. Reson. 1987; 73: 375-379Google Scholar). 15NT 1 values were measured from spectra recorded with eight different durations of the delay: 44.4, 111.1, 222.2, 333.2, 555.4, 777.6, 999.7, and 1444.0 ms for CBFβ(141) and 44.4, 111.1, 222.2, 333.2, 444.3, 555.4, 666.5, 888.6, and 1333.0 ms for CBFβ(187). 15N T 2 values were measured from spectra recorded with delays corresponding to the duration of the CPMG sequence: 16.3, 32.6, 48.9, 65.2, 81.4, 97.7, 130.3, and 162.9 ms for CBFβ(141) and 16.3, 32.6, 48.9, 65.2, 81.4, 97.7, and 114.0 ms for CBFβ(187). To generate pure absorptive 2D line shapes, the N- and P-type signals recorded for each t 1 point were stored separately to carry out the necessary addition and subtraction of free induction decay and 90 ° phase correction as described previously (36Kay L.E. Keifer P. Saarinern T. J. Am. Chem. Soc. 1992; 114: 10663-10665Crossref Scopus (2432) Google Scholar). The necessary data manipulations were carried out using software written in-house. All other data processing was carried out using the program PROSA (39Güntert P. Dötsch V. Wider G. Wüthrich K. J. Biomol. NMR. 1992; 2: 619-629Crossref Scopus (279) Google Scholar). The intensities of the peaks in the two-dimensional spectra were analyzed by measuring the peak heights using the integration routine in the program XEASY (40Bartels C. Xia T.-H. Billeter M. Güntert P. Wüthrich K. J. Biomol. NMR. 1995; 5: 1-10Crossref PubMed Scopus (1604) Google Scholar). The uncertainties in the measured peak intensities were set equal to the root-mean-square base-line noise of the spectra. The T 1 andT 2 values were determined by fitting the measured peak heights to the following two-parameter function:I(t)=I0exp−tT1,2Equation 1 in which I(t) is the intensity at timet and I 0 is the intensity at time zero. T 1 and T 2 values and uncertainties were determined by nonlinear least squares fitting of the experimental data points to the monoexponential decay given in Equation 1 using the Levenburg-Marquardt algorithm (41Press W.H. Flannery B.P. Teukolsky S.A. Vetterling W.T. Numerical Recipes. Cambridge University Press, Cambridge1986: 683-688Google Scholar) to minimize the χ2 goodness of fit parameter. The goodness-of-fit of the data to Equation 1 was evaluated by comparison of the calculated χ2 value with tabulated values of χ2 at 95% confidence level as described previously (42Palmer III, A.G. Rance M. Wright P.E. J. Am. Chem. Soc. 1991; 113: 4371-4380Crossref Scopus (597) Google Scholar). An initial estimate of the correlation time for CBFβ(141) and CBFβ(187) were obtained from a 10% trimmed mean of theT 1/T 2 ratio (43Kay L.E. Torchia D.A. Bax A. Biochemistry. 1989; 28: 8972-8979Crossref PubMed Scopus (1794) Google Scholar) for those backbone amides with T 1 andT 2 values deemed adequate by the goodness-of-fit analysis described above. The biophysical and structural studies we wish to carry out to characterize CBFβ require large quantities of homogeneous protein. Attempts to overexpress CBFβ in a number of different vectors including glutathione S-transferase fusion vector" @default.
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