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- Results and Discussion
- Experimental Section
Materials: All N-α-Fmoc-protected amino acids were purchased from Nova Biochem (Darmstadt, Germany), GL Biochem (Shanghai, China), Fluka (Taufkirchen, Germany), Bachem (Bubendorf, Switzerland), Merck (Darmstadt, Germany), and IRIS Biotech (Marktredwitz, Germany). All other chemicals were purchased from Sigma–Aldrich (Taufkirchen, Germany), ABCR (Karlsruhe, Germany), Acros Organics (Geel, Belgium), Alfa Aesar (Karlsruhe, Germany), New England Biolabs (Ipswich, USA), Macherey&Nagel (Düren, Germany). Preloaded Gly or Lys-p-benzyloxybenzyl alcohol resin (Wang resin, 0.3 mmol g−1), N-hydroxybenzotriazole (HOBt), O-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluorophosphate (HBTU) were purchased from GL Biochem (Shanghai, China). N,N′-Diisopropylcarbodiimide (DIC) and N-ethyldiisopropylamine (DIPEA) were obtained from IRIS Biotech (Marktredwitz, Germany). 5(6)-Carboxyfluoresceine (CF), 2-mercaptoethane sulfonate sodium (MESNa) and N-methyl-2-pyrrolidinon (NMP) were purchased from Fluka (Taufkirchen, Germany). Trifluoroacetic acid (TFA) was bought from Roth (Karlsruhe, Germany).
ESI-MS: Data were obtained with a Finnigan instrument (type LCQ) or Bruker spectrometers (types Apex-Q IV 7T, HCT ultra, and micrOTOF API). High-resolution (HR) spectra were obtained with the Bruker Apex-Q IV 7T or the Bruker micrOTOF.
RP-HPLC: All RP-HPLC analyses were performed on instruments from GE Healthcare or Jasco. For all semi-preparative/preparative RP-HPLC, we used a Pharmacia Äkta basic device (GE Healthcare) with a gradient of eluent A (0.1 % TFA in H2O) to eluent B (0.1 % TFA in MeCN/H2O 8:2) with a flow rate of 3 mL min−1 (semipreparative)/10 mL min−1 (preparative) were performed. Preparative purification was performed on a Phenomenex column Jupiter, RP-C18, 250×20 mm, 5 μm, 80 Å. For semi-preparative purification, a Phenomenex column Jupiter, RP-C18, 250×10 mm, 5 μm, 80 Å was used. For analytical RP-HPLC, a semi-micro-HPLC system from Jasco was used, applying a gradient of eluent A (0.1 % TFA in H2O) to eluent B (0.1 % TFA in MeCN) with a flow rate of 1 mL min−1. In these cases, a Phenomenex column RP-C18, 250×4.6 mm, 5 μm was used. UV detection was performed at 215 nm, 254 nm and, for the fluorophore-labeled peptides, at 444 nm.
Circular dichroism (CD) spectroscopy: CD spectra were recorded on a Jasco-810 spectropolarimeter equipped with a Jasco PTC432S temperature controller. Prior to usage, the sample cell was flushed with nitrogen. For CD spectra recording zinc-finger derivatives, 1 mm quartz glass precision cells were used. The peptide concentrations were adjusted to between 5–30 μM for all derivatives. The spectra were recorded at 20 °C in a wavelength range of 260–190 nm for the zinc fingers and from 300–200 nm for the DNA binding studies with 1.0 nm bandwidth in continuous mode, 1.0 s response and a scan speed of 100 nm min−1. Eight spectra were averaged. Spectra were background-corrected, smoothed (Savitzky–Golay), and depicted as molar ellipticity (Θ ×10−5 deg cm2 dmol−1).
Cloning of Zf12: The cDNA encoding the murine transcription factor Zif268 (EgrI) was purchased from ATCC (Wesel, Germany). After amplifying the desired DNA fragment of the two zinc-finger domains (632–827) by polymerase chain reaction (PCR) using the forward 5‘GGTGGTGCTAGCGAACGCCCATATGCTTGCCCTG3’ and reverse 5‘GGTGGTTGCTCTTCCGCAGTCCTTCTGTCTTAAATGGATTTTG3’ primers from Sigma–Aldrich (Taufkirchen, Germany), and purification by agarose-gel electrophoresis, the fragment was ligated into the cloning vector pGEM-T from Promega (Mannheim, Germany). After digestion of the cloning vector pGEM-T and expression vector pTXB1 from New England Biolabs (Ipswich, USA), using the restriction enzymes SapI and NheI, the DNA insert and vector were subsequently ligated. To confirm an in-frame cloning of Zf12 (1), DNA sequencing was performed.
Protein expression of Zf12 in E. coli: E. coli ER2566 cells were transformed with the pTXB1/Zf12 (632–827) DNA plasmid and grown in lysogeny broth (LB) or 2*YT medium containing carbenicilline (100 μg mL−1). A preparatory culture (5 mL) was used to inoculate the expression culture. The cells were cultivated at 200 rpm at 37 °C, until the culture reached an optical density at 600 nm (OD600) of 0.6–0.8. The culture was then induced by adding 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubated again overnight at 16 °C. The cells were harvested by centrifugation at 4 °C at 9000 g and lysed in buffer A: 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic (HEPES) buffer (20 mm), NaCl (500 mm), tris(2-carboxyethyl)phosphine (0.1 mm), Tween20 (0.1 %), pH 8, and 20 μM phenylmethylsulfonylfluoride (PMSF) was added before incubating on ice for 30 min. Complete lysis was accomplished by pulsed sonication (5×45 s). The soluble extracts of the protein were isolated by centrifugation at 23 000 g for 20 min at 4 °C. Purification and isolation of Zf12 (1) was enabled by an attached chitin binding domain (CBD) as follows: a column (GE Healthcare, XK 26/20) filled with 100 mL chitin beads (New England Biolabs) was equilibrated at 4 °C with water (2×bed volumes) and buffer A (10×bed volumes). Loading of the cell lysate proceeded overnight at a flow rate of 1 mL min−1 (10 times). Unspecifically bound E. coli proteins were removed by washing with buffer A (3×bed volumes). The intein fusion protein of Zf12 was cleaved in the presence of buffer B: HEPES (20 mm), MESNa (250 mM, pH 6) over 70 h at 4 °C. The peptide thioester was eluted with buffer A. The complete procedure was monitored at 4 °C by UV absorption (280 nm, Äkta Prime Plus, GE Healthcare). Analysis of the peptide thioester was carried out by SDS-PAGE (tris-tricine 10–20 %), RP-HPLC on a C18 semi-preparative column (Phenomenex column Jupiter RP-C18, 250×10 mm, 5 μm, 80 Å) with 0.1 % TFA in H2O (A) and 0.1 % TFA in H2O/MeCN (20:80) (B) as eluting system with a 2050 % gradient of B over 30 min at a flow rate of 3 mL min−1, and HRMS-ESI.
Reduced Zf12 (1): H2N-ASERPYACPVESCDRRFSRSDELTRHIRIHTGQKPFQCRICMRNFSRSDHLTTHIRTHTGEKPFAOH; Mr=7794.8 (C328H520N110O98S7); MS (ESI): m/z=650.57 [M+12H]12+, 709.62 [M+11H]11+, 780.48 [M+10H]10+; HRMS-ESI: m/z calcd for C328H520N110O98S7: 709.3348 [M+11H]11+, found: 709.3460; 780.1786 [M+10H]10+, found: 780.1788.
SPPS of Zf3 and respective modifications: The peptides were synthesized by standard Fmoc/tert-butyl peptide synthesis on solid support on preloaded Wang resin, 0.3 mmol g−1 using a microwave-supported automated peptide synthesizer LibertyTM (CEM Cooperation, Matthews, NC, USA). The side chain protection groups were tert-butyl for aspartic acid, glutamic acid, serine, tyrosine; tert-butyloxycarbonyl (Boc) or allyloxycarbonyl (alloc) for lysine; triphenylmethyl (trityl) for cysteine, glutamine and histidine; and 2,2,4,6,7-pentamethyl-dihydrofurane-5-sulfonyl (PBF) for arginine. Resins were swollen in CH2Cl2 for 30 min. The coupling protocol was adjusted to the different needs of the amino acids coupled. Fmoc amino acids were used as 0.2 M solutions in N-methylpyrrolidone (NMP). Coupling was performed with 0.5 M HBTU/HOBt (5 equiv) in DMF, 0.2 m amino acids in NMP (5 equiv) and 2 m DIPEA (10 equiv) in NMP. Double coupling of the amino acids arginine, cysteine and aspartic acid was performed to enhance the coupling efficiency. Capping was performed after every coupling cycle using a solution of acetic anhydride (10 %), DIPEA (5 %), and HOBt (0.2 %) in NMP. All reaction steps were performed under microwave conditions (20 W, 300 s at 75 °C) and N2 mixing. Arginine was coupled under N2 mixing (1500 s) at RT followed by microwave-assisted coupling (300 s) at 75 °C. To minimize racemization during coupling, cysteine derivatives were coupled for 300 s at 50 °C. Histidine was coupled without using the microwave supply for 60 min.23 The Fmoc-protecting group was removed by two deprotection cycles with 20 % piperidine in NMP (30 s, 180 s). Final deprotection of the Fmoc group was performed for all peptides except the orthogonal Lys(alloc)-protected derivatives required for further fluorophore coupling. Prior to cleavage, the resins were washed with DMF/CH2Cl2 (1:1, 5×5 mL), CH2Cl2 (3×5 mL), EtOH (3×5 mL), and CH2Cl2 (5×5 mL) and dried overnight. Cleavage from the resin and simultaneous removal of all side chain protecting groups was performed separately for all derivatives in a 10 mL syringe (Becton, Dickinson & Co., Franklin Lakes, NJ, USA) by adding TFA/H2O/triethylsilyl/1,2-ethanedithiol (v/v/v/v=95:2:1:2, 10 mL g−1 resin) and shaking for 2 h at RT. The peptides were precipitated from ice-cold tert-butylmethylether, isolated by centrifugation (9000 rpm, 4 °C) and washed with ice-cold Et2O (3×); the pellet was subsequently lyophilized from H2O using a freeze dryer (Martin Christ GmbH, Osterode am Harz, Germany). The lyophilized peptides were purified by semi-preparative/preparative RP-HPLC on C18 columns (Phenomenex column Jupiter RP-C18, 250×10 mm, 5 μm, 80 Å with 0.1 % TFA in H2O (A) and 0.1 % TFA in H2O/MeCN (20:80) (B) as eluting system with a 20–50 % gradient of B over 30 min at a flow rate of 3/10 mL min−1 and lyophilized again. The peptides were analyzed by HRMS-ESI.
Analysis of Zf3 and its modifications: The purified reduced peptides were analyzed by RP-HPLC on C18 semi-preparative column (Phenomenex column RP-C18, 250×10 mm, 5 μm), with 0.1 % TFA in H2O (A) and 0.1 % TFA in H2O/MeCN (20:80) (B) as eluting system at a flow rate of 3 mL min−1, and HRMS-ESI.
Ligation of Zf13 and its modifications: The N-terminal Cys 65 of the different Zf3 peptides had to be available for nucleophilic attack at the thioester of 1 during the ligation step. Therefore, the disulfide bridges in Zf3 and the other constructs were first reduced. The peptide (3 mM) was dissolved in phosphate buffer (10 mM, pH 4, TCEP 20 mM) and stirred overnight at RT under inert conditions. Complete reduction of the peptide was confirmed by ESI-MS. Subsequently, peptide thioester 1 (1 mM) was added in phosphate buffer (500 mM, pH 8) and the pH of the solution was adjusted to pH 7.8–8. As determined by RP-HPLC, the conversion of the two peptides into the ligation product was observed after stirring for 6–12 h at RT under inert conditions.
Analysis of the ligation products: The ligation products were analyzed by RP-HPLC on an analytical C18 column (Phenomenex column RP-C18, 250×4.6 mm, 5 μm) with 0.1 % TFA in H2O (A) and 0.1 % TFA in MeCN (B) as eluting system with a 20–50 gradient of B over 30 min at a flow rate of 1 mL min−1 and ESI-MS.
Zf13 A85: H2NASERPYACPVESCDRRFSRSDELTRHIRIHTGQKPFQCRICMRNFSRSDHLTTHIRTHTGEKPFACDICGRKFARSDERKRHTKIALRQKDGOH; Mr=10 843.4 (C458H741N159O134S7); RP-HPLC: gradient B 2040 % over 40 min, flow rate=0.3 mL min−1, tR=27.5 min; MS (ESI): m/z=603.3 [M+18H]18+, 678.4 [M+16H]16+, 723.5 [M+15H]15+, 834.8 [M+13H]13+, 904.4 [M+12H]12+, 986.7 [M+11H]11+, 1085.2 [M+10H]10+, 1205.7 [M+9H]9+, 1401.1 [M+8H]8+, 1356.3 [M+7H]7+; HRMS: m/z calcd for C458H741N159O134S7: 678.3456 [M+16H]16+, found: 678.3536; calcd: 1085.248 [M+10H]10+, found: 1085.237; calcd: 1205.720 [M+9H]9+, found: 1205.703; calcd: 1356.309 [M+7H]7+, found: 1356.291.
ZF13 A85 L89: H2NASERPYACPVESCDRRFSRSDELTRHIRIHTGQKPFQCRICMRNFSRSDHLTTHIRTHTGEKPFACDICGRKFARSDERKRHTKIALRQK(FL)DGOH; Mr=11 215.5 (C480H753N159O140S7); RP-HPLC: gradient B: 2050 % over 30 min, tR=18.62 min; MS (ESI): m/z=701.3 [M+16H]16+, 747.9 [M+15H]15+, 801.3 [M+14H]14+, 862.8 [M+13H]13+, 934.6 [M+12H]12+, 1019.5 [M+11H]11+, 1121.2 [M+10H]10+, 1245.6 [M+9H]9+, 1401.1 [M+8H]8+, 1601.1 [M+7H]7+, 1867.6 [M+6H]6+.
ZF13 TACN85: H2NASERPYACPVESCDRRFSRSDELTRHIRIHTGQKPFQCRICMRNFSRSDHLTTHIRTHTGEKPFACDICGRKFARSDERKRHTKITACNLRQKOH; Mr=10 893.5 (C462H751N163O130S7); RP-HPLC: gradient B: 2050 % over 30 min, tR=18.9 min; MS (ESI): m/z=574.5 [M+19H]19+, 606.4 [M+18H]18+, 642.0 [M+17H]17+, 682.0 [M+16H]16+, 727.4 [M+15H]15+, 779.3 [M+14H]14+, 891.1 [M+13H]13+, 908.9 [M+12H]12+, 991.4 [M+11H]11+, 1090.4 [M+10H]10+.
ZF13 IDAO70: H2NASERPYACPVESCDRRFSRSDELTRHIRIHTGQKPFQCRICMRNFSRSDHLTTHIRTHTGEKPFACDICGIDAOKFARSDERKRHTKIHLRQKOH; Mr=10 811.4 (C458H737N157O134S7); RP-HPLC: gradient B: 2050 % over 30 min, tR=18.74 min; MS (ESI): m/z=570.2 [M+19H]19+, 601.8 [M+18H]18+, 637.1 [M+17H]17+, 676.9 [M+16H]16+, 721.9 [M+15H]15+, 773.4 [M+14H]14+, 832.8 [M+13H]13+, 902.1 [M+12H]12+, 983.9 [M+11H]11+, 1082.2 [M+10H]10+.