Synthesis of Leucine Zippers and Leucine Zipper Dendrimers

Transcription

1 Supplementary Materials Helical Supramolecules and Fibers Utilizing Leucine-Zipper Displaying Dendrimers Min Zhou, David Bentley, Indraneel Ghosh Contribution from the Department of Chemistry, University of Arizona, Tucson, Arizona Synthesis of Leucine Zippers and Leucine Zipper Dendrimers General Materials. All amino acid derivatives, HOBT, BOP and resins used for peptide synthesis were purchased from Novabiochem. All other chemicals and reagents were from Aldrich. HPLC columns were from Vydac. Peptide Synthesis and Purification: The peptide-ez and peptide-kz were synthesized by solid-phase peptide synthesis using standard Fluorenylmethoxycarbonyl (Fmoc) strategy starting with Fmoc-Cys(Trt)-Wang resin ( mesh). Sequences: EZ: NH 2 -AQALEKELQALEKELQALEWELQALEKELSGSGC-COOH KZ: NH 2 -AQALKKKLQALKKKLQALKWKLQALKKKLSGSGC-COOH The final peptides were cleaved from the resin and all protecting groups were removed by the general TFA cleavage protocol. Peptide resin 2.1g was treated with 1.5ml cleavage cocktail (94.5% TFA, 2.5% water, 2.5 Ethanedithiol and 1% triisopropylsilane) for 90 minutes at room temperature. The solution was filtered and precipitated by transferring into a centrifuge tube containing 20 ml of cold ether. After centrifugation and 3 x washes with fresh ether, the white solid was collected and lyophilized to yield 0.8 g crude peptide. The crude peptides were subsequently purified by reverse-phase HPLC (Varian) using a C18 column (Vydac). Peptides were typically purified with a linear gradient of 20% to 80% acetonitrile containing 0.1% TFA at a flow rate of 10ml/min, for 60 minutes. Mass Spectroscopy (MALDI) of the two peptides: The MH + calculated for peptide-ez is ; found: The MH + calculated for peptide-kz is ; found Amino Acid Analysis Peptide-EZ: Glx (12) 12.22, Ser (2) 1.64, Gly (2) 2.04, Ala (5) 5.00, Leu (8) 8.06, Lys (3) Peptide-KZ: Glx (4) 3.95, Ser (2) 1.69, Gly (2) 1.54, Ala (5) 5.00, Leu (8) 7.99, Lys (11) The results are consistent with the desired primary sequences. Synthesis of Succinimido 3-Maleimidopropanoate (1) The procedure was essentially as decribed in Nicholas. 1 β-alanine (0.91g, 10mmol) was added to a solution of maleic anhydride (1.0g, 10mmol) in 12ml DMF and stirred for 2 hours at room temperature. When the solid dissolved, an ice bath was used to decrease the temperature to 0 ºC. 1 Nicholas J. Ede, et. al., Bioconjugate Chem., 1994, 5, S1

2 N-Hydroxysuccinimide (1.44g, 12.5mmol) was added into the solution followed by DCC (4.12g, 20mmol). After 5-10 minutes the ice bath was removed and the reaction was kept at room temperature overnight, which resulted in a white precipitate. The solution was filtered and the precipitate was washed with 60ml water and 60ml dichloromethane. The filtrate was collected and the organic layer was washed with 3 X 20ml 5% NaHCO 3 and saturated NaCl solution. The organic layer was dried with Na 2 SO 4 and the dichloromethane removed under reduced pressure. 1.6g solid (60% yield) was obtained. Synthesis of maleimide derivatized PAMAM dendrimer, generation 0 (2) 0.5 g of 20 % w/w of (0.07 mmol) PAMAM dendrimer, generation 0 (Aldrich) in methanol was added into a round-bottom flask and methanol was removed under reduced pressure. 1ml DMSO was added into the flask to dissolve the dendrimer. Then 0.2 g of (1) (0.75 mmol) was dissolved in 1 ml DMSO to which was added 0.1 g (0.78 mmol) of diisopropylethylamine. After 2 minutes, the activated ester was added into the flask containing the dendrimer solution. The flask was kept shaking at room temperature for 30 minutes. The resullting red solution was acidified by acetic acid and purified by reverse-phase HPLC on a C18 column. 22 mg of pure product (2) was obtained. ESI analysis confirmed the identity of the product. Mass Spectroscopy (ESI) calculated for compound (2) is ; Found: H NMR (DMSO): δ 2.3 (t, 8H, J=7.5Hz), 2.48 (m, 12H), 3.04 (m, 24H), 3.58 (t, 8H, J=7.5Hz), 6.99 (s, 8H), 8.00 (s, 4H), 8.12 (s, 4H). Figure S1. Pink ball used in Fig. 1 of manuscript represents the maleimide modified PAMAM generation 0 dendrimer. The peptides EZ and KZ and their sequences are also shown below. S2

3 Synthesis of Dendrimer (D-EZ 4 ) and (D-KZ 4 ) D-EZ 4 was synthesized starting with 33.6 mg of purified peptide-ez dissolved in 800µl buffer (triethylamine/acetic acid, ph = 7). 0.5 mg of 2 (0.42 µmol) was added to the peptide-ez solution. Either triethylamine or acetic acid was used to adjust the ph of this solution (ph 6-7). The reaction was kept overnight at 37 ºC. The product, D-EZ 4, was purified by reverse-phase HPLC on a C18 column using a linear gradient of triethylamine/acetic acid (ph=7) and CH 3 CN, 10% to 60%, at a flow rate of 10ml/min, for 60 minutes. 2.5 mg of pure D-EZ 4 was obtained and characterized by MALDI (see below). D-KZ 4 was synthesized essentially as above. D-KZ 4 was purified by reverse-phase HPLC on a C18 column with a linear gradient of 20% to 50% acetonitrile containing 0.1% TFA at a flow rate of 10ml/min, for 60 minutes. 2 mg of pure D-KZ 4 was obtained starting from 40 mg of purified peptide-kz. Reinjection of both D-EZ 4 and DKZ 4 onto a C18 analytical column verified that they were greater than 95% pure. Mass Spectroscopy (MALDI) of the D-EZ 4 and DKZ 4 : MALDI calculated for D-EZ 4 (M + Na + ) is Found MALDI calculated for D-KZ 4 (M + Na + ) is Found: A CPK model of the leucine-zipper dendrimer is shown below. S3

4 Circular Dichroism (CD) Spectroscopy: EZ and KZ Controls Experimental Circular Dichroism spectra were recorded on an Aviv 62A-DS spectropolarimeter. All measurements reported were carried out in 10 mm phosphate buffer at the appropriate ph, using a cuvette with a 1mm pathlength. Peptide and dendrimer-peptide hybrid concentrations were determined from the Tryptophan absorbance at 280 nm (6 M Gdn.HCl) and independently verified by Amino-acid analysis. All CD experiments were conducted at 25 o C and care was taken to minimize peptide exposure to atmosphere to prevent disulfide bond formation. Results and Discussion EZ and KZ ph titrations In order to establish the feasibility of monitoring the complexation of DEZ 4 /4KZ and DKZ 4 /4EZ (Fig 2a & 2b) we carried out a ph titration of the peptides EZ and KZ, to choose an appropriate ph regime where only one of the two peptide species was fully folded and helical. The results are shown in Fig. S2 where EZ is helical below ph 5.8 and KZ is helical above ph 7.8 Figure S2. ph titration of EZ( )and KZ ( )as monitored by the change in helicity at 222 nm and expressed as fraction folded. The concentration of EZ and KZ were 20 µm in 10 mm phosphate at the indicated ph. S4

5 EZ titrated with KZ at ph 8.4 and KZ titrated with EZ at ph 5.6 The ph 8.4 and ph 5.6 buffer was chosen for further studies as the CD spectra of the folded peptides, KZ at ph 8.4 and EZ at ph 5.6, could be subtracted from that of the EZ/KZ complexes allowing us to selectively monitor the change in secondary structure of the unstructured leucine zippers as a function of increasing concentration of the folded leucine zipper. In order to verify that the experiments were valid for EZ and KZ we titrated 20 µm EZ with varying concentrations of KZ at ph 8.4 (Fig S3a) and we also titrated 20 µm KZ with varying concentrations of EZ at ph 5.6 (Fig. S3b). The study clearly established the change in helicity of EZ as a function of KZ at ph 8.4 was due to the formation of 1:1 complexes of EZ/KZ. Similarly it was also established that KZ and EZ formed 1:1 complexes at ph 5.6. This control study was essential in allowing us to validate the complexation experiments for D-EZ 4 with KZ at ph 8.4 and D-KZ 4 with EZ at ph 5.6 respectively. a) b) ph 8.4 ph 5.6 Figure S3. CD spectra of EZ titrated with KZ and KZ titrated with EZ at ph 8.4 and ph 5.6 respectively. (a) Subtraction CD spectra of 20 µm EZ in the presence ( ) and absence ( ) of KZ (20 µm); (b) Subtraction CD spectra of 20 µm KZ in the presence ( ) and absence ( ) of EZ (20 µm). The insets show the average (±10% error) of three titrations of 20 µm EZ ( ) and 20 µm KZ ( ) with increasing concentrations of KZ and EZ respectively. S5

6 FT-IR Spectroscopy Experimental Solution FT-IR spectra of EZ/KZ (pd 7.0), D-KZ 4 /4EZ (pd 5.6 ), and D-EZ 4 /4KZ (pd 8.4) and the β-sheet (pd 7.0) control (KLVFFAE )were acquired in 10 mm phosphate in D 2 O in a CaF 2 cell with a 0.05 mm pathlength in a Nicolet Avatar 360 FTIR (0.5 cm-1) at a total concentration of 1.0 mg/ml. For D-EZ 4 /D-KZ 4, the complex (0.78 mg/ml) was allowed to precipitate over 16 hours in 10 mm phosphate at pd 7.0 before acquiring spectra as above. The peak at 1673 cm -1 in our samples corresponds to trace levels of TFA salts from HPLC purification. Figure S4. FT-IR Spectra of complexes. D-EZ 4 /D-KZ 4 fiber (green) overlayed upon the solution FT-IR spectra of D-KZ 4 /4EZ (blue), D-EZ 4 /4KZ (black) and EZ/KZ (cyan) complexes. The beta-sheet (Bsheet) control is included as a reference Results and Discussion The solution spectra of the different leucine-zipper based complexes (D-EZ 4 /4 KZ, D-KZ 4 /4EZ and EZ/KZ) display almost identical FT-IR spectra, with Amide I peaks centered at 1635 cm -1 and 1645 cm -1. The 1635 cm -1 and 1645 cm -1 centered Amide I peaks are characteristic of leucine-zippers. What is most interesting is that the spectra of the fibrillar sample, DEZ 4 /DKZ 4 (green) was very similar to that of the other leucine-zipper complexes, characterized by CD and FT-IR, which clearly suggests that the fiber retains the intended helical secondary structure. In order to clearly exclude any significant beta-sheet structure in our complex, we also acquired the spectra of a fragment (KLVFFAE) of the beta-amyloid protein known to adopt a beta-sheet structure. The control beta-sheet peptide shows an Amide I peak centered at 1620 cm -1 where our D-EZ 4 /D-KZ 4 fiber does not significantly absorb. These experiments lead us to conclude that the fibers formed from the D-EZ 4 /D-KZ 4 have helical secondary structure characteristic of coiled-coil assemblies. 1 1 Heimburg, T.; Schunemann, J.; Weber, K.; Geisler, N.; FTIR-spectroscopy of multistranded coiled coil proteins, Biochemistry, 1999, 38, S6

7 Sedimentation Equilibrium Analysis Materials and Methods Sedimentation Equilibrium data 1 was collected for three concentrations (20 µm, 40 µm and 60 µm) of DKZ 4 /4EZ, DKZ 4 and EZ at ph 5.6 and DEZ 4 /4KZ, DEZ 4 and KZ at ph 8.4 as well as three rotor speeds 10000, and RPM on a Beckman Optima XL-I ultracentrifuge. All samples were in 10 mm phosphate and 1 mm DTT at the appropriate ph after extensively dialyzing the solutions for 12 hours. Equlibrium at each speed was judged to be complete when three consecutive scans taken at two-hour intervals were indistinguishable from each other. Typical equilibrium experiments at each speed were carried out over a period of 24 hours at a particular rotor speed. Data was analyzed using SEDEQ 4.1 (Allen Minton, Laboratory of Biochemical Pharmacology, NIH). Results and Discussion The results of the sedimentation equilibrium experiments are tabulated below and representative data are shown in Figures S5 and S6. ph Sample Actual Mol. Wt. Observed Mol. Wt. 5.6 Table S1. Sedimentation Equilibrium Data D-KZ 4 + 4EZ 31,367 Da 30,510 Da EZ 3,785 Da 14,310 Da tetramer: 15,140 Da D-KZ 4 16,227 Da 19,080 Da 8.4 D-EZ 4 + 4KZ 31,384 Da 30,700 Da KZ 3,776 Da 14,720 Da tetramer: 15,104 Da D-EZ 4 16,280 Da 18,270 Da We utilized distinct ph regimes, either ph 8.4 (for D-EZ 4 /KZ) or ph 5.6 (for D-KZ 4 /EZ) to clearly show that only the desired complexes are present by conducting control sedimentation equilibrium experiments with only D-EZ 4, D-KZ 4, EZ and KZ at the appropriate phs. These experiments clearly show that only the DEZ 4 /4KZ (ph 8.4) and the DKZ 4 /4EZ (ph 5.6) form complexes of 31 kd (31.3 kd theoretical) with very good distribution of residuals (Fig. S5a and Fig. S6a). We have also established that D-EZ 4 (ph 8.4) and D-KZ 4 (ph 5.6) both exist as monomers of 18 kd and 19 kd (16.3 kd theoretical) (Fig. S5e and Fig. S6e), whereas the 1 Laue, T. M.; Shah, B. D.; Ridgeway, T. M.; Pelletier, S. L. in Analytical Ultracentrifugation in Biochemistry and Polymer Science; Harding, S. E., Rowe, A. J., Horton, J. C., Eds.; The Royal Society of Chemistry: Cambridge, S7

8 unmodified peptides, EZ (ph 5.6) and KZ (ph 8.4) exist as tetramers (Fig. S5d and Fig. S6d). as previously observed for a very similar leucine-zipper peptide sequence by Lumb & Kim, Biochemistry, 1995, 34, 8642 In order to exclude any ambiguity in our results, we also show the fits of both D-KZ 4 /EZ and D-EZ 4 /KZ to a molecular weight of 15 kd (Fig. S5b and Fig. S6b). The lack of a good fit is obvious, indicating that this does not agree with the data. Furthermore, we also allowed the data to include the possibility of different fractions (f1 and f2) of two species (M1 and M2) for both D-EZ 4 /KZ and D-KZ 4 /EZ, one at 31 kd (M1) and the other at 16 kd (M2) (Fig. S5c and Fig. S6c). This leads to a result where there is only M1 present and essentially no M2 (less than 1% as seen by the value for f2) for both D-EZ 4 /KZ and D-KZ 4 /EZ. Thus, these results clearly establish that we form the proposed complexes, DEZ 4 /4KZ (ph 8.4) and the DKZ 4 /4EZ (ph 5.6), which is in agreement with the CD titration data. a) b) c) d) e) Figure S5. Shows the sedimentation Equilibrium data at ph 5.6 in 10 mm phosphate and 1 mm DTT. Fits are shown for a) D-KZ 4 /4EZ b) D-KZ 4 /4EZ fit to 15 kd; c) D-KZ 4 /4EZ fit to two species M1*f1 + M2*f2; d) EZ; and e) D-KZ 4 S8

9 a) b) c) d) e) Figure S6. Shows the Sedimentation Equilibrium data at ph 8.4 in 10 mm phosphate and 1 mm DTT. Fits are shown for a) D-EZ 4 /4KZ b) D-EZ 4 /4KZ force fit to 15 kd; c) D- EZ 4 /4KZ fit to two species M1*f1 + M2*f2; d) KZ; and e) D-EZ 4 S9

10 Transmission Electron Microscopy (TEM) Materials and Methods The samples (5µM of D-EZ 4 and D-KZ 4 incubated 8-12 hrs) were mixed with 2% phosphotungstic acid, ph adjusted to 7.2 with sodium hydroxide in a 1:1 ratio. The samples were prepared in two different methods 1 : a) The mixture was placed onto the surface of a previously Piloform plastic, carbon coated (60-90nm plastic, approximately 5nm carbon) (Hayat, 2000, pg 227) 150 mesh grid and allowed to stand for 10 minutes. Excess fluid was withdrawn with a fine pipette tip and the samples allowed to dry. (Hayat, 2000, pg 371) b) Alternatively, the samples were mixed with stain as above, but placed onto the surface of a previously Piloform plastic, carbon coated (60-90nm plastic, approximately 5nm carbon) (Hayat, 2000, pg 227) 150 mesh grid and allowed to stand for 10 minutes. Excess fluid was withdrawn with a fine pipette tip and the samples were allowed to dry. (Hayat, 2000, pg 371). The samples were observed and photographed in a JEOL 100 CX II TEM at 80 kv at various levels of magnification. Additional TEM images are shown in accompanying supplementary materials. 1 Hayat, M.A. in Electron Microscopy; Biological Applications, Fourth edition. Cambridge University Press, New York S10