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• W2061640372 abstract "Antitumor antibiotic chromoproteins such as neocarzinostatin involve a labile toxin that is tightly bound by a protective protein with very high affinity but must also be freed to exert its function. Contrary to the prevalent concept of ligand release, we established that toxin release from neocarzinostatin requires no major backbone conformational changes. We report, herein, that subtle changes in the side chains of specific amino acid residues are adequate to gate the release of chromophore. A recombinant wild type aponeocarzinostatin and its variants mutated around the opening of the chromophore binding cleft are employed to identify specific side chains likely to affect chromophore release. Preliminary, biophysical characterization of mutant apoproteins by circular dichroism and thermal denaturation indicate that the fundamental structural characteristics of wild type protein are conserved in these mutants. The chromophore reconstitution studies further show that all mutants are able to bind chromophore efficiently with similar complex structures. NMR studies on 15N-labeled mutants also suggest the intactness of binding pocket structure. Kinetic studies of chromophore release monitored by time course fluorescence and quantitative high pressure liquid chromatography analyses show that the ligand release rate is significantly enhanced only in Phe78 mutants. The extent of DNA cleavage in vitro corresponds well to the rate of chromophore release. The results provide the first clear-cut indication of how toxin release can be controlled by a specific side chain of a carrier protein. Antitumor antibiotic chromoproteins such as neocarzinostatin involve a labile toxin that is tightly bound by a protective protein with very high affinity but must also be freed to exert its function. Contrary to the prevalent concept of ligand release, we established that toxin release from neocarzinostatin requires no major backbone conformational changes. We report, herein, that subtle changes in the side chains of specific amino acid residues are adequate to gate the release of chromophore. A recombinant wild type aponeocarzinostatin and its variants mutated around the opening of the chromophore binding cleft are employed to identify specific side chains likely to affect chromophore release. Preliminary, biophysical characterization of mutant apoproteins by circular dichroism and thermal denaturation indicate that the fundamental structural characteristics of wild type protein are conserved in these mutants. The chromophore reconstitution studies further show that all mutants are able to bind chromophore efficiently with similar complex structures. NMR studies on 15N-labeled mutants also suggest the intactness of binding pocket structure. Kinetic studies of chromophore release monitored by time course fluorescence and quantitative high pressure liquid chromatography analyses show that the ligand release rate is significantly enhanced only in Phe78 mutants. The extent of DNA cleavage in vitro corresponds well to the rate of chromophore release. The results provide the first clear-cut indication of how toxin release can be controlled by a specific side chain of a carrier protein. The search for stable and nontoxic carrier-based drug delivery systems is a new trend in the recently emerging field of ligand-targeted therapeutics (1Allen T.M. Cullis P.R. Science. 2004; 303: 1818-1822Crossref PubMed Scopus (3711) Google Scholar). The protein-mediated ligand-selective and high affinity carriers offer a promising strategy in drug targeting and controlled delivery of small therapeutic chemical agents (2de Wolf F.A. Brett G.M. Pharmacol. Rev. 2000; 52: 207-236PubMed Google Scholar). In light of the rapid advancements, understanding the naturally ingenious ways by which the bioactive ligands are released and controlled has been the subject of substantial interest, especially among biological chemists. The mechanism of noncovalent ligand release has been studied for many carrier proteins. Lipoprotein receptor family, α-tocopherol transfer protein, retinol-binding proteins, pheromone-binding proteins, etc., are a few examples. Most models of ligand release from carrier protein complexes are hitherto found to be concomitant or subsequent to significant conformational changes in the protein structure (see Refs. 3Beglova N. Blacklow S.C. Trends Biochem. Sci. 2005; 30: 309-317Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 4Leal W.S. Chen A.M. Ishida Y. Chiang V.P. Erickson M.L. Morgan T.I. Tsuruda J.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 5386-5391Crossref PubMed Scopus (150) Google Scholar, 5Min K.C. Kovall R.A. Hendrickson W.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14713-14718Crossref PubMed Scopus (120) Google Scholar for a few examples; see Refs. 6Koshland D.E. Science. 1963; 142: 1533-1541Crossref PubMed Scopus (123) Google Scholar, 7Kempner E.S. FEBS Lett. 1993; 326: 4-10Crossref PubMed Scopus (86) Google Scholar, 8Gerstein M. Krebs W. Nucleic Acids Res. 1998; 26: 4280-4290Crossref PubMed Scopus (313) Google Scholar for reviews and data base). The enediyne family of antitumor antibiotics (9Shen B. Liu W. Nonaka K. Curr. Med. Chem. 2003; 10: 2317-2325Crossref PubMed Scopus (74) Google Scholar, 10Xi Z. Goldberg I.H. Barton D.S. Nakanishi K. Comprehensive Natural Products Chemistry. Vol. 7. Elsevier, New York1999: 553-592Crossref Google Scholar) includes some of the most potent antitumor agents and appears to be the only well studied category of the natural antibiotic chromoproteins. Neocarzinostatin (NCS) 2The abbreviations used are: NCS, neocarzinostatin; holoNCS, holoneocarzinostatin; apoNCS, aponeocarzinostatin; NCS-C, NCS chromophore; WT, wild type; HPLC, high pressure liquid chromatography; HSQC, heteronuclear single quantum coherence; EK, enterokinase; CBP, calmodulin-binding peptide. (secreted by Streptomyces carzinostaticus), being the first member of this family (11Ishida N. Miyazaki K. Kumagai K. Rikimaru M. J. Antibiot. (Tokyo) Ser. A. 1965; 18: 68-76PubMed Google Scholar), has intrigued scientists by virtue of its novel architecture, unusually high DNA cleavage activity, and antitumor effect (12Abe S. Otsuki M. Curr. Med. Chem. Anti-Cancer Agents. 2002; 2: 715-726Crossref PubMed Scopus (44) Google Scholar). The protein component of NCS, aponeocarzinostatin (apoNCS), is biologically inactive but plays important roles in protecting and transferring the labile chromophore (NCS-C) to the target DNA (13Goldberg I.H. Acc. Chem. Res. 1991; 24: 191-198Crossref Scopus (338) Google Scholar). It is interesting to note that apoNCS does not bind to DNA (14Jung G. Kohnlein W. Biochem. Biophys. Res. Commun. 1981; 98: 176-183Crossref PubMed Scopus (22) Google Scholar), and its biological activity relies on the release of the tightly bound NCS-C (Kd ≤ 0.1 nm) (15Povirk L.F. Goldberg I.H. Biochemistry. 1980; 19: 4773-4780Crossref PubMed Scopus (93) Google Scholar) before NCS interacts with DNA. The fundamental mechanism underlying such a process displays a fascinating partnership between the biologically active chromophore and the apoprotein-based drug delivery system. apoNCS is an all-β protein with 113 amino acid residues and two disulfide bonds. It possesses a deep cavity for noncovalent binding of its chromophore (16Gao X. J. Mol. Biol. 1992; 225: 125-135Crossref PubMed Scopus (17) Google Scholar, 17Teplyakov A. Obmolova G. Wilson K. Kuromizu K. Eur. J. Biochem. 1993; 213: 737-741Crossref PubMed Scopus (38) Google Scholar, 18Kim K.H. Kwon B.M. Myers A.G. Rees D.C. Science. 1993; 262: 1042-1046Crossref PubMed Scopus (83) Google Scholar). The crystal structures of apoNCS and holoNCS are reported to be almost superimposable (18Kim K.H. Kwon B.M. Myers A.G. Rees D.C. Science. 1993; 262: 1042-1046Crossref PubMed Scopus (83) Google Scholar). The striking similarity between the structures of apoNCS and holoNCS imply that distinct structural changes might not be necessary for the binding or release of NCS-C. We also found that chromophore release of holoNCS can precede major secondary and/or tertiary structural changes in the protein (19Sudhahar G.C. Balamurugan K. Chin D.-H. J. Biol. Chem. 2000; 275: 39900-39906Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). This leads to a logical speculation of whether and which, if any, side chains can control the release via subtle fluctuations in protein conformation. We anticipate that the possible candidates responsible for gating the chromophore release should satisfy two criteria. First, they must reside on the surface rather than the interior of the protein. Second, they are likely to be located in the opening of the binding cleft rather than being distributed in other parts of the surface. Physiological release of a noncovalently bound ligand from a protein complex is often triggered by a specific contact with some trans-acting agents in a cell. Such contact, if present, is more likely to occur on the solvent-exposed surface than the interior. Next, when backbone conformation remains unchanged, association or dissociation of a ligand must be through the opening of the binding cleft. Fig. 1 illustrates an aqueous model of holoNCS (20Chin D.-H. Chem.-Eur. J. 1999; 5: 1084-1090Crossref Scopus (17) Google Scholar) adapted from the crystal structure (pdb.1nco.ent) (see supplementary Fig. S1 on line for the stereo view of holoNCS complex). The NCS-C is clamped in between two β-hairpin loops, designated as Loop 1 (between strands 7 and 8) and Loop 2 (between strands 9 and 10). NMR studies on dynamics and relaxation of NCS reveal internal motion and localized flexibility in the loop regions around the binding cleft (21Mispelter J. Lefevre C. Adjadj E. Quiniou E. Favaudon V. J. Biomol. NMR. 1995; 5: 233-244Crossref PubMed Scopus (33) Google Scholar, 22Valerio-Lepiniec M. Nicaise M. Adjadj E. Minard P. Desmadril M. Protein Eng. 2002; 15: 861-869Crossref PubMed Scopus (11) Google Scholar). We speculate that these loop residues (residues 75–80 in Loop 1 and residues 98–102 in Loop 2) may play a pivotal role and thus set up mutagenesis studies to explore the effect of these side chains on chromophore release. Despite large efforts in the past to clone apoNCS, it was reported only in recent years by a few groups using a synthesized artificial gene (23Heyd B. Lerat G. Adjadj E. Minard P. Desmadril M. J. Bacteriol. 2000; 182: 1812-1818Crossref PubMed Scopus (30) Google Scholar, 24Nozaki S. Tomioka Y. Hishinuma T. Inoue M. Nagumo Y. Tsuruta L.R. Hayashi K. Matsumoto T. Kato Y. Ishiwata S. Itoh K. Suzuki T. Hirama M. Mizugaki M. J. Biochem. (Tokyo). 2002; 131: 729-738Crossref PubMed Scopus (14) Google Scholar, 25Urbaniak M.D. Muskett F.W. Finucane M.D. Caddick S. Woolfson D.N. Biochemistry. 2002; 41: 11731-11739Crossref PubMed Scopus (36) Google Scholar). We report for the first time a construct carrying the natural apoNCS gene and apply it for the first time in an enediyne toxin release study. Not willing to compromise on any possible loss of the structural identity of recombinant apoNCS to that of natural one from S. carzinostaticus in our mutagenesis studies, we designated a convenient strategy to produce recombinant apoNCS identical to the natural one, including at the termini. We also carefully performed a disulfide assay to ascertain the formation of the disulfide linkage in the recombinant apoNCS. Such concern could not be overlooked because our study highly depends on the quality of the folded conformation. Extraction of NCS-C—The NCS powder was from Kayaku Co., Ltd. (Itabashi-Ku, Tokyo, Japan). Repeated methanolic extraction of NCS-C was carried out from lyophilized NCS stock (0.5 mm) in 5 mm sodium citrate, pH 4.0, following the described method (26Kuo H.-M. Lee Chao P.-D. Chin D.-H. Biochemistry. 2002; 41: 897-905Crossref PubMed Scopus (11) Google Scholar). The integrity and concentration of NCS-C were checked by HPLC analysis and the increased A340 (ε, 10,800) after titrating with excess apoNCS. Reconstitution of holoNCS—The reconstitution of holoNCSs was performed in ice by adding an equimolar (1:1) amount of the methanol-extracted NCS-C into a solution of recombinant protein in 5 mm ammonium acetate, pH 4.0. The mixture was incubated at 0 °C for 5–10 min to ensure complete reconstitution. The holoNCSs were freshly made before experiments, and the methanol amount in the reaction mixture was adjusted to below 5%. Care was taken to avoid degradation of the labile chromophore, and the integrity of the NCS-C after reconstitution was examined by HPLC analysis. pCAL-n-EK-apoNCS Construct—The genomic DNA template was isolated from vegetative cultures of S. carzinostaticus (ATCC 15944). The sequence of the ncsA gene (GenBank™, National Institutes of Health genetic sequence data base (gi|420324|) (27Sakata N. Minamitani S. Kanbe T. Hori M. Hamada M. Edo K. Biol. Pharm. Bull. 1993; 16: 26-28Crossref PubMed Scopus (10) Google Scholar), which encodes the 147-amino acid pre-apoNCS with a signal peptide at the N terminus, was used for primer design. The native gene of the 113-amino acid apoNCS was amplified by PCR with the following primers: forward, 5′-GACGACGACAAGGCGGCGCCGACGGCTACGGT-3′; and reverse, 5′-GGAACAAGACCCGTCGGAGCGGATCCTCCGATCA-3′. The 12-nucleotide upstream and 13-nucleotide downstream flanking sequences were included for ligation-independent cloning in T7/lac promoter-based Escherichia coli expression vector, pCAL-n-EK (Stratagene, La Jolla, CA). The vector (50 ng), after it is digested with Eam11041 and gel-purified, was incubated at 72 °C for 10 min in a solution containing 1 mm dTTP and Pfu DNA polymerase (ligation-independent cloning kit; Stratagene, La Jolla, CA) to produce 12- and 13-nucleotide ligation-independent cloning single-stranded overhangs. Similarly, 175 ng of the gel-purified PCR amplicons was treated with Pfu DNA polymerase in the presence of 1 mm dATP. The vector and insert were annealed at room temperature for 1 h of incubation, and the product was transformed into E. coli DH5α. The apoNCS gene was fused downstream to the sequence encoding the N-terminal calmodulin affinity peptide (CBP) tag that was combined with the EK cleavage site (28Wyborski D.L. Bauer J.C. Zheng C.F. Felts K. Vaillancourt P. Protein Expression Purif. 1999; 16: 1-10Crossref PubMed Scopus (13) Google Scholar, 29Vaillancourt P. Zheng C.F. Hoang D.Q. Briester L. Methods Enzymol. 2000; 326: 340-362Crossref PubMed Google Scholar). The inclusion of the EK site allowed complete removal of the CBP tag and generated apoNCS with the native N terminus (i.e. Ala1 in apoNCS). The DNA sequencing of PCR amplicons and pCAL-n-EK-apoNCS construct was performed twice on automated ABI prism sequencer model 310 (Applied Biosystems, CA) to verify the correctness of the sequence. Site-specific Mutant Constructs of apoNCS—A PCR-based quick change mutagenesis method was applied to obtain site-specific mutant constructs of apoNCS. The codon GCG and CUG were used for the replacement to Ala and Leu, respectively, according to the codon usage of E. coli (30Nakamura Y. Wada K. Wada Y. Doi H. Kanaya S. Gojobori T. Ikemura T. Nucleic Acids Res. 1996; 24: 214-215Crossref PubMed Scopus (55) Google Scholar). Followed by PCR, DpnI was used to deplete the unmutagenized DNA. The mutant construct was transformed into E. coli DH5α, and the constructed plasmids were sequenced at least twice until the correct sequence was repeatedly confirmed. Expression and Purification of Recombinant apoNCS Proteins—The pCAL-n-EK expression construct of native or mutated apoNCSs was transformed into E. coli BL21 Codon Plus (Stratagene, La Jolla, CA). The expression of CBP-apoNCS fusion protein was induced by 0.2 mm isopropyl β-d-thiogalactopyranoside at 30 °C for 5 h. After ultrasonication of the isopropyl β-d-thiogalactopyranoside-induced E. coli cells, the CBP-apoNCS fusion protein was found in soluble fraction of the cell extract. The CBP-apoNCS fusion protein was purified using calmodulin affinity resin (Stratagene, La Jolla, CA) (Stratagene, La Jolla, CA) (28Wyborski D.L. Bauer J.C. Zheng C.F. Felts K. Vaillancourt P. Protein Expression Purif. 1999; 16: 1-10Crossref PubMed Scopus (13) Google Scholar, 29Vaillancourt P. Zheng C.F. Hoang D.Q. Briester L. Methods Enzymol. 2000; 326: 340-362Crossref PubMed Google Scholar). The protein was subsequently desalted and concentrated through Amicon or Centricon cellulose membrane (MWCO: 3000) (Millipore, Bedford, MA). The quantity of the recovered CBP-apoNCS fusion protein was evaluated by UV spectrophotometry. The extinction coefficient of the fusion protein at 278 nm, ε278 = 21,400 m–1, was estimated from the reported value for apoNCS, ε278 = 14,400 m–1 (31Povirk L.F. Dattagupta N. Warf B.C. Goldberg I.H. Biochemistry. 1981; 20: 4007-4014Crossref PubMed Scopus (91) Google Scholar, 32Napier M.A. Holmquist B. Strydom D.J. Goldberg I.H. Biochem. Biophys. Res. Commun. 1979; 89: 635-642Crossref PubMed Scopus (176) Google Scholar), and the calculated value for the CBP tag protein, ε278 = 7000 m–1 (ProtParam) (33Gasteiger E. Hoogland C. Gattiker A. Duvaud S. Wilkins M.R. Appel R.D. Bairoch A. Walker J.M. The Proteomics Protocols Handbook. Humana Press, Totowa, NJ2005: 571-607Crossref Google Scholar). For each mg of the CBP-apoNCS fusion protein, 1 unit of EK (Invitrogen, CA, or GenScript, Piscataway, NJ) was added, and the solution was incubated at room temperature for several days. When the enzymatic cleaving of the CBP tag was more than 90% complete (checked by SDS-PAGE), the mixture was separated by DEAE-Sepharose (Fast Flow) resin (Amersham Biosciences AB, Uppsala, Sweden) with a linear gradient of 0–400 mm NaCl. The final protein product was collected after dialysis against water or filtration through Amicon or Centricon cellulose membrane (MWCO: 3000). The purified protein was analyzed by SDS-PAGE and HPLC. The averaged purity was greater than 95%. The authenticity of each protein was verified by mass spectrometry and disulfide linkage test, as described below. The final yield of recombinant apoNCS and mutants was at a range of 0.25–4 mg/liter of LB culture. Aggregation was frequently observed during purification of wild type (WT) recombinant apoNCS and mutants. This caused low yield from some mutants. For instance, the average yield of D79A was only 0.25 mg/liter of LB culture. Among all mutants purified for the study, F78L showed the highest yield (4 mg/liter of LB culture). Expression and Purification of 15N-Labeled Proteins—Uniform 15N labeling was achieved by growing the host bacteria, E. coli BL21 Codon Plus, with vitamin B1 in M9 minimal medium containing 0.25 g/liter of 15NH4Cl (Cambridge Isotope Laboratories, Inc., MA) as the sole nitrogen source. The purification of the labeled apoNCS and mutants was carried out following the same procedure as described above. The yield was about half of that of the corresponding unlabeled protein. Changing ammonium chloride from 0.50 to 0.125 g/liter of medium did not significantly reduce or improve the protein yield. Mass Spectrometry—All of the mass spectrometries were done on a Finnigan LCQ mass spectrometry detector (Thermo Electron, San Jose, CA) equipped with atmospheric pressure ionization source using electrospray ionization (+)-charge mode. The protein samples were diluted in 30% acetonitrile containing 0.1% trifluoroacetic acid for analysis. NMR Spectroscopy—One-dimensional 1H NMR spectra of unlabeled WT apoNCS and mutants were recorded at room temperature on a Varian 600-MHz NMR spectrometer (Palo Alto, CA). The two-dimensional 15N-1H heteronuclear single quantum coherence (HSQC) NMR spectra were recorded on a Bruker DMX 600-MHz spectrometer (Rheinstetten, Germany) at 25 °C. The NMR samples were prepared by dissolving lyophilized protein in 20 mm sodium phosphate, pH 7.0, in 10% D2O, 90% H2O. The cross-peaks for WT apoNCS were identified based on the reported assignments (25Urbaniak M.D. Muskett F.W. Finucane M.D. Caddick S. Woolfson D.N. Biochemistry. 2002; 41: 11731-11739Crossref PubMed Scopus (36) Google Scholar). The cross-peak of Tyr32, which is in a tyrosine corner-like motif and is considered to contribute stability of apoNCS (34Nicaise M. Valerio-Lepiniec M. Izadi-Pruneyre N. Adjadj E. Minard P. Desmadril M. Protein Eng. 2003; 16: 733-738Crossref PubMed Scopus (9) Google Scholar), was used here as an internal reference. CD Spectroscopy—All of the CD measurements were carried out on a Jasco J-715 spectropolarimeter (Tokyo, Japan) equipped with a circulating water bath (Neslab, model RTE-140) (Portsmouth, NH). For apoNCS and its mutants, a 200-μl protein solution in 20 mm sodium phosphate, pH 7.0, with a concentration level of 10–15 μm for far UV CD and 50 μm for near UV CD, was used for measurement. The spectrum was recorded at 25 °C using a temperature controlled water-jacketed cell with 0.1-cm-path length at a scan speed of 20 nm/min. For reconstituted WT and mutated holoNCSs, a 10 μm sample in 20 mm ammonium acetate, pH 4.0, was used for measurement. Because of the instability of holoNCSs, the scan speed was increased to 100 nm/min to reduce time. All of the spectra were accumulated minimum five times and are corrected for the respective buffer blanks. The data are expressed as the mean residue ellipticity for both apoNCS and holoNCS (19Sudhahar G.C. Balamurugan K. Chin D.-H. J. Biol. Chem. 2000; 275: 39900-39906Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Fluorescence Spectroscopy—The fluorescent changes from a holoNCS sample in kinetic release study were monitored by a SLM Aminco Bowman Series II luminescence spectrometer (SLM Aminco Bowman, Urbana, IL). The sample temperature was controlled at 25 °C by a thermostatic cell holder equipped with a refrigerated thermostatic bath circulator. The sample of volume 150 μl in a 3-mm square cell was used. A single point acquisition of emission data at 440 nm was collected once per 30 s by excitation at 340 nm. To avoid the degradation of holoNCSs by excessive exposure to the light source, the light shutter was controlled by modification of time scan mode using an in-house written macro language commands so that it can be closed in between data collections. In a 100-min monitoring, exposure of NCS to light was minimized to 2.33 min. A rapid kinetics setup was used for fast releasing mutant F78L, where the shutter control was suspended for the initial 3 min to collect data once per 5 s. After that, the shutter control was enabled as described. Disulfide Linkage Test—A facile disulfide evaluation procedure was developed for checking the integrality of the produced recombinant apoNCS and its mutants. To 100 μl of solution of 10 μm apoNCS protein in 0.1 m sodium phosphate, pH 8.0, guanidine hydrochloride was added to a final concentration of 4 m. After mixing, 80-fold molar excess of iodoacetamide over apoNCS (20-fold excess/Cys residue) was added at 37 °C for 2 h. Under this treatment, alkylation can occur on any un-oxidized Cys residues in apoNCS. The protein sample was then desalted and analyzed by mass spectrometry. Thermal-induced Denaturation—The thermal-induced denaturationof50 μm of WT apoNCS and mutants, in 10 mm sodium phosphate buffer, pH 7, was monitored by CD spectroscopy at 224 nm from 25–91 °C with 3 °C increments. The temperature was controlled by a microprocessor and a temperature sensor. The equilibrium time was 15 min, and the ellipticity was recorded for 30 s at each temperature setting. Kinetic Study on the Release of NCS-C—The rate of NCS-C release from natural, reconstituted WT or mutated holoNCSs was determined using the method previously published (19Sudhahar G.C. Balamurugan K. Chin D.-H. J. Biol. Chem. 2000; 275: 39900-39906Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The reconstituted holoNCSs (10 μm) were prepared in 100 mm Tris-HCl, pH 7.0, and the release of NCS-C was monitored by fluorescence spectroscopy at 25 °C after the addition of 5 mm GSH. The final reading of the emission was obtained after the sample was treated with 80% isopropanol (to complete the release of NCS-C). The release kinetics was also independently examined by HPLC analyses. The samples (0.5 nmol), prepared as above, were drawn at different time points and were analyzed by reverse phase HPLC. The quantity of the intact NCS-C and GSH-induced adduct, which represent population of the protein-bound and released species, respectively, were estimated using the method described (35Chin D.-H. Tseng M.-C. Chuang T.-C. Hong M.-C. Biochim. Biophys. Acta. 1997; 1336: 43-50Crossref PubMed Scopus (11) Google Scholar). Kinetic Release Data Processing Information—The rate of release is expressed as d[holoNCS]/dt=−kobs[holoNCS]u=−k1[holoNCS] (Eq. 1) where kobs and n are the observed rate constant and reaction order for the rate of disappearance of holoNCS, respectively, and k1 is the first order rate constant after approximation. However, the release rate decreased gradually with time when ratio of apoNCS to NCS-C increased with progressing lose of the intact NCS-C. For consistency, all of the rate constants were calculated from the initial 10% release that fit into a first order linear plot (the averaged r = 0.97 analyzed by the Kaleidagraph program (Synergy Software, Reading, PA)). holoNCS-induced DNA Damage—The DNA cleavage ability of each chromoprotein complex was assessed by the proportion of conversion from form I DNA (supercoiled) to form II (relaxed; caused by single strand breaks) and form III (linear; caused by double-stranded breaks). The supercoiled pBR322 DNA was isolated from E. coli using Mini-Prep plasmid isolation kit (Qiagen Inc.). A 16-μl DNA drug reaction mixture was prepared by mixing 5 μm (final concentration) of freshly reconstituted holoNCS, 5 mm GSH, 100 mm Tris-HCl, pH 7.0, and 40 ng/μlof pBR322 DNA. The mixture was incubated at 16 °C for 15 min and was immediately loaded onto a 1% w/v agarose gel for analysis. HPLC Analyses—When release kinetics was followed by HPLC, the amount of the protein bound (intact form) and the released NCS-C (inactivated by GSH into an adduct) were analyzed by a Waters Millennium HPLC equipped with a 600E solvent delivery system, a 996 photodiode array detector, and a Waters 474 or a Jasco FP-1520 fluorescence detector using the described method (19Sudhahar G.C. Balamurugan K. Chin D.-H. J. Biol. Chem. 2000; 275: 39900-39906Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 35Chin D.-H. Tseng M.-C. Chuang T.-C. Hong M.-C. Biochim. Biophys. Acta. 1997; 1336: 43-50Crossref PubMed Scopus (11) Google Scholar). The purity of WT and mutated apoNCSs (0.5–1 nmol) were examined using a gradient that starts with a sharp increase from 0 to 30% of CH3CN (containing 0.1% trifluoroacetic acid) in 2 min, followed by a shallow linear increase to 35 and 40% in 17 and 52 min, respectively. The chromophore separation involved the gradient that starts with a sharp increase from 0 to 48% of acidic methanol containing 5 mm ammonium acetate at pH 5, within the initial 3 min, followed by a shallow linear increase to 80% in 65 min. Cloning and Characterization of Recombinant apoNCS Proteins—After a long attempt, we successfully isolated the native gene of the 113-amino acid apoNCS from S. carzinostaticus. The use of pCAL-n-EK allowed expression of a fusion protein from which apoNCS could be retrieved by a proteolytic removal of its tag by EK. The recombinant apoNCS thus obtained possessed the native termini without any extraneous residues. After DEAE-Sepharose ion exchange chromatography, the purified recombinant apoNCS and mutants showed a single band corresponding to the band of the natural apoNCS in SDS-PAGE. A single peak was observed by HPLC analysis, indicating the homogeneity of the purified proteins. The mono isotopic mass number of the recombinant WT apoNCS (11085 ± 1), as determined by electrospray ionization-mass spectrometry measurement, matches with the theoretical value (11085.2). The mono isotopic mass numbers of all mutants and 15N-labeled proteins are also in good agreement with their theoretical molecular weights. Disulfide Assay—To avoid any possible artifacts, it was crucial to examine whether all disulfide linkages were properly formed in the produced proteins. This assay was specifically developed for apoNCS, and the sensitivity and validity have been evaluated and confirmed. 3C.-J. Tseng and D.-H. Chin, unpublished data. As evidenced by mass spectrometry measurement, no alkylated species was observed for all purified proteins obtained in the current investigation. The results demonstrate the absence of free–SH in cysteine residues and indicate that the WT and all mutated apoNCSs contain two disulfides as the natural one does. CD Spectra of WT and Mutated apoNCS—The structural characterization of WT and mutant apoNCSs was carried out by CD. The far UV CD spectrum of the recombinant WT apoNCS (Fig. 2A) revealed a predominant β-sheet structure. The profile is consistent with that reported for the native apoNCS (19Sudhahar G.C. Balamurugan K. Chin D.-H. J. Biol. Chem. 2000; 275: 39900-39906Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 23Heyd B. Lerat G. Adjadj E. Minard P. Desmadril M. J. Bacteriol. 2000; 182: 1812-1818Crossref PubMed Scopus (30) Google Scholar, 36Napier M.A. Holmquist B. Strydom D.J. Goldberg I.H. Biochemistry. 1981; 20: 5602-5608Crossref PubMed Scopus (41) Google Scholar, 37Jayachithra K. Kumar T.K.S. Lu T.-J. Yu C. Chin D.-H. Biophys. J. 2005; 88: 4252-4261Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). The far UV CD spectra of F78A, F78L, D79A, and S98A mutants also closely resembled that of WT apoNCS. Only a slight deviation was observed in the spectra of F76A and D99A mutants. Overall, the characteristic f" @default.
• W2061640372 created "2016-06-24" @default.
• W2061640372 creator A5029711136 @default.
• W2061640372 creator A5037435278 @default.
• W2061640372 creator A5046331643 @default.
• W2061640372 creator A5057448553 @default.
• W2061640372 date "2006-06-01" @default.
• W2061640372 modified "2023-10-18" @default.
• W2061640372 title "A New Model for Ligand Release" @default.
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