Biomacromolecules 2008, 9, 2111–2117
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New Design of Helix Bundle Peptide-Polymer Conjugates Jessica Y. Shu,† Cen Tan,† William F. DeGrado,‡ and Ting Xu*,†,§,| Department of Materials Science and Engineering, University of California, Berkeley, California 94720, Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, Department of Chemistry, University of California, Berkeley, California 94720, Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 Received February 1, 2008; Revised Manuscript Received May 27, 2008
We present a new design of peptide-polymer conjugates where a polymer chain is covalently linked to the side chain of a helix bundle-forming peptide. The effect of conjugated polymer chains on the peptide structure was examined using a de novo designed three-helix bundle and a photoactive four-helix bundle. Upon attachment of poly(ethylene glycol) to the exterior of the coiled-coil helix bundle, the peptide secondary structure was stabilized and the tertiary structure, that is, the coiled-coil helix bundle, was retained. When a heme-binding peptide as an example is used, the new peptide-polymer conjugate architecture also preserves the built-in functionalities within the interior of the helix bundle. It is expected that the conjugated polymer chains act to mediate the interactions between the helix bundle and its external environment. Thus, this new peptide-polymer conjugate design strategy may open new avenues to macroscopically assemble the helix bundles and may enable them to function in nonbiological environments.
Introduction Peptide-polymer conjugates have the potential to combine the advantages of synthetic polymers, peptides, and peptidomimetics and can lead to hierarchically ordered, functional soft materials.1–12 Various types of peptide-polymer conjugates have been designed and investigated. Most are linear di- or triblock copolymers and can be divided into two families based on the complexity of the peptide sequences and the specificity of the intra- and interpeptide interactions. The first family consists of an amino acid homopolymer as one block and either a synthetic polymer or polypeptide as the second block. The peptide homopolymer typically forms secondary structures and, upon phase separation of the block copolymer, assembles within microdomains, resulting in hierarchical assemblies with subnanometer features.6,10,11 Although specific interactions, such as electrostatic interactions and hydrogen bonding, have been introduced between each block to direct and manipulate the conjugate assemblies, the peptide’s built-in functionalities associated with a unique sequence are lost. The second family of peptide-polymer conjugates contains a specific peptide sequence with tailored intra- and interpeptide interactions. Peptides forming R-helices and β-sheets, as well as cyclic peptides, have been used as building blocks.1,4,5,7–9,13–19 With these, hierarchical assemblies having molecular level control over chemical heterogeneity have been achieved. Furthermore, peptides forming tertiary structures, such as the leucine zipper, have also been used as physical cross-linking points to generate hydrogels for tissue engineering.7,8,20 There have been, however, limited studies on peptide-polymer conjugates that utilize the built-in functionalities of helix bundles to achieve high selectivity, specificity or responsiveness to external stimuli, as observed in natural proteins. * To whom correspondence should be addressed. E-mail: tingxu@ berkeley.edu. † Department of Materials Science and Engineering. ‡ Department of Biochemistry and Biophysics. § Department of Chemistry. | Lawrence Berkeley National Laboratory.
Coiled-coil helix bundles, found in more than 200 natural proteins, underpin many structural and catalytic functions of natural proteins, such as enzymatic activity, signal transfer and redox chemistry. Recently, a large number of peptide sequences forming helix bundles have been de novo designed (designed with novel amino acid sequences) to not only mimic natural protein functionalities, but also to perform functions not seen in nature.21–30 These peptides have both chemical and structural diversity that are comparable to or greater than those found in natural materials and are typically more robust.31 They are, however, limited by stability and degradation encountered during handling and are not amenable to standard fabrication processes that generate technologically important functional materials. Upon conjugating synthetic polymers to the helix bundle and obtaining proper assemblies, the synthetic polymers could act to protect, deliver and template the peptides on the nanometer scale, while the peptide bundles could provide molecular level control over the chemical heterogeneity to trigger, direct, and execute built-in functionalities.1,3,32 The success in achieving desirable, functional assemblies relies on fundamentally understanding the interactions between each building block and delicately balancing and manipulating these interactions to achieve targeted assemblies without interfering with helix bundle formation or designed functionalities. We have designed and studied a new family of helix bundleforming peptide-polymer conjugates, with the polymer chain covalently linked to the peptide side chain. Upon attaching poly(ethylene glycol) (PEG) chains to the exterior of a previously de novo designed three-helix bundle, the peptide secondary structure is stabilized. Also, the presence of PEG does not interfere with the peptide tertiary structure, that is, the coiledcoil helix bundle. More importantly, using a photoactive, hemebinding, four-helix bundle-forming peptide as an example, this new design preserves the built-in functionalities within the interiorofthehelixbundle.Thisdesignstrategyforpeptide-polymer conjugates opens a new avenue toward generating functional biomaterials that use more sophisticated peptide structures to achieve high selectivity, sensitivity and responsiveness, as seen
10.1021/bm800113g CCC: $40.75 2008 American Chemical Society Published on Web 07/16/2008
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Biomacromolecules, Vol. 9, No. 8, 2008
in nature. With the unique architecture of this designed peptide-polymer conjugate, the peptide structure is retained and both termini of the peptide are accessible, making it feasible to generate hydrogels with tailored spatial distributions and aggregation states of chemical motifs for tissue engineering. More importantly, the polymer chains on the exterior of the helix bundle could provide a handle to mediate interactions with the external environment, could potentially enable the macroscopic self-assembly of the helix bundles, and could allow the helix bundle-based machineries to function in nonbiological environments. With the chemical and structural diversity of either naturally existing or de novo designed helix bundles and the many advantages of synthetic polymers, the prospect of fabricating materials with novel properties, superior to natural materials, is clearly possible.
Materials and Methods Materials. Two peptides, referred hereafter as 1CW (Ac-EVEALEKKVAALESKVQALEKKVEALEHG WDGR-CONH2) and H10H24 (Ac-GGGEIWKLHEEFLKKFEELLKLHEERLKKM-CONH2) were investigated.33,34 The peptides were synthesized on an AAPPTEC Apex 396 solid phase synthesizer using standard 9-fluorenylmethyl carbamate (Fmoc) protection chemistry on Wang resin (Nova Biochem), typically at 0.3 mmol scale. The side chain protecting groups were as follows: Lys(Boc), Glu(OtBu), Asp(OtBu), Cys(Trt), Arg(Pbf), His(Trt), Trp(Boc), Ser(tBu), Gln(Trt). For the synthesis of 1CW-PEG conjugates, the serine at position 14 was mutated to cysteine to facilitate conjugation of maleimide end-functionalized PEG. Similarly for H10H24, the lysine at position 15 was mutated to cysteine. Prior to peptide cleavage from the resin, the N-terminus was acetylated using a 1:1 (v/v) acetic anhydride: pyridine solution for 30 min. The peptides were cleaved from the resin and simultaneously deprotected using 90:8:2 trifluoroacetic acid (TFA)/ethanediol/water for 3.5 h. Crude peptides were precipitated in cold ether and subsequently dissolved in water and lyophilized. Maleimide end-functionalized PEG, purchased from Rapp Polymere (Germany), was then coupled to the cysteine residues of the peptides, which were in white powder form, in 25 mM potassium phosphate buffer at pH 8 for 1 h.3 PEGs of three varying molecular weights were utilized: 750, 2000, and 5000 Da. These are referred henceforth as PEG750, PEG2K, and PEG5K, respectively. Reversed-Phase High-Pressure Liquid Chromatography (RPHPLC). Peptides and their conjugates were purified by RP-HPLC (Beckman Coulter) on a C18 column (Vydac). Samples were eluted with a linear AB gradient, where solvent A consisted of water plus 0.1% (v/v) TFA and solvent B consisted of acetonitrile plus 0.1% (v/ v) TFA. For purification of 1CW and its conjugates, the linear AB gradient of 37 to 42% B over 25 min was used, with typical elution of 1CW between 38-39% B and that of 1CW-PEG750, 1CW-PEG2K, and 1CW-PEG5K between 40-41% B. For purification of H10H24, typical elution was ∼42% B on a gradient from 41 to 45% B over 20 min. H10H24-PEG2K and H10H24-PEG5K eluted at ∼39% B on a 30 to 40% B gradient over 30 min. Peptide elution was monitored with a diode array detector at wavelengths of 220 and 280 nm. The flow rate was 10 mL/min for semipreparative runs and peptides were injected at a concentration of 10-20 mg/mL. MALDI-TOF Mass Spectrometry. The identity and purity of the peptides were verified by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry using R-cyano-4hydroxycinnamic acid matrix. Mass spectra were recorded on an Applied BioSystems Voyager-DE Pro. UV-Vis. Peptides were dissolved in buffered aqueous solution containing 25 mM potassium phosphate (KH2PO4) at pH 8 and 100 mM potassium chloride (KCl). Spectra were recorded on a HewlettPackard 8453 spectrophotometer using a standard 1 cm path length quartz cuvette. Peptide concentrations in solution were determined by their absorption at 280 nm due to each peptide’s lone tryptophan residue,
Shu et al. assuming an extinction coefficient of 5500 M-1 cm-1, and using the Beer-Lambert Law. Titration experiments were done using UV-vis spectroscopy with various concentrations of H10H24, H10H24-PEG2K, and H10H24PEG5K solutions in quartz cuvettes. Spectra were recorded after addition of each aliquot, either 0.5 or 1 µL, depending on the concentration of hemin solution in DMSO, typically 200-500 µM, and the concentration of the peptide solution. The dissociation constant, Kd, of each heme binding site was determined by monitoring the shift of the heme absorbance at 412 nm as a function of the ratio of heme to four-helix bundles and assuming extinction coefficients of 120000 M-1 cm-1 for bound heme and 35000 M-1 cm-1 for free heme.22 Kd was calculated as Kd ) [A][B]/[AB], where [A] is the concentration of free peptide in solution, [B] is the concentration of free heme in solution, and [AB] is the concentration of heme bound to H10H24. This specific titration experiment was repeated four times to ensure reproducibility. Detailed descriptions of the evaluation of the heme dissociation constants can be found in the Supporting Information. Circular Dichroism. Circular dichroism measurements for secondary structure characterization were made on a Jasco J810 spectropolarimeter. CD spectra of each sample were recorded three times and averaged by collecting data from 260 to 190 at 0.2 nm intervals, a rate of 20 nm/ min, a response time of 4 s, and a bandwidth of 1 nm. Samples were dissolved in 25 mM KH2PO4, 100 mM KCl buffer of various pH (3, 5, 8, 11). 1 mm path length quartz cuvettes were used for peptide concentrations g10 µM, and 2 cm cuvettes were used for concentrations
