Hybrid Hydrogels Cross-Linked by Genetically Engineered Coiled

coiled-coil protein structure and stability on hydrogel volume transition, a series of ... interspersed naturally derived recombinant coiled-coils was...
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Biomacromolecules 2001, 2, 912-920

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Hybrid Hydrogels Cross-Linked by Genetically Engineered Coiled-Coil Block Proteins Chun Wang,† Jindrˇich Kopecˇ ek,†,‡ and Russell J. Stewart*,† Departments of Bioengineering and Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84112 Received March 9, 2001; Revised Manuscript Received May 17, 2001

Hybrid hydrogels of hydrophilic synthetic polymers cross-linked by protein modules undergo externally triggered volume transitions as a result of protein conformational changes. To investigate the influence of coiled-coil protein structure and stability on hydrogel volume transition, a series of block proteins containing interspersed naturally derived recombinant coiled-coils was synthesized. Proteins were characterized using circular dichroism, size exclusion chromatography, gel electrophoresis, and analytical ultracentrifugation. The block proteins formed self-associating oligomers and displayed thermal unfolding profiles indicative of a hierarchic higher-order structure. Hybrid hydrogels were assembled from an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer and His-tagged block proteins through metal complexation. A temperature-induced decrease in hydrogel swelling was observed, and the onset temperature of the volume transition corresponded to the onset temperature of protein unfolding. We conclude that stimuli-responsive properties of hybrid hydrogels can be tailored by engineering the structure and properties of protein crosslinks. Introduction Stimuli-responsive hydrogels are cross-linked networks of hydrophilic polymer chains that undergo volume transitions induced by changes in environmental conditions. At any equilibrium state, swelling of hydrogels due to solvation of polymer chains is balanced by the elastic force exerted by the polymer network immobilized through cross-links. Volume transitions occur when this balance of forces is perturbed, which may be due to changes in the polymersolvent interaction parameter, disruption/formation of crosslinks, or order-disorder transition of polymer domains. Stimuli-responsive hydrogels have been proposed for a variety of applications.1,2 Numerous types of synthetic stimuli-responsive hydrogels have been reported that responded to temperature,3,4 pH,5,6 solvent,7 electric and magnetic fields,8,9 light,10 and biochemicals.11,12 The best studied systems are based on a limited number of chemically synthesized copolymers of N-isopropylacrylamide (temperature-sensitive), (meth)acrylates (pH-sensitive), and their derivatives. Stimuli-responsive hydrogels have also been prepared from recombinant proteins and polypeptides.13-15 Recently, two-component hybrid hydrogels have been developed that have novel environmental response mechanisms. Generally, one of the components is a hydrophilic synthetic polymer, and the other is a molecule of biological origin that functions as chemical or physical cross-link. Biomolecule cross-links include oligopeptide sequences recognized by specific proteases,16-18 full-length native * To whom correspondence should be addressed at: [email protected]. † Department of Bioengineering, University of Utah. ‡ Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah.

proteins and their ligands such as glucose-concanavalin A19 and antibody-antigen complexes,20 complementary strands of oligodeoxyribonucleotides,21 and oligo(glycolic acid)s with complementary D/L conformations.22 Stimuli response of the hybrid hydrogels is largely determined by the biological component. Recombinant protein domains have also been used as cross-links in hybrid hydrogels.23-25 In these gels the “microscopic” structural sensitivity of protein domains was exploited to control “macroscopic” volume transitions of hydrogels. Domains are structurally well-defined and independently folded building blocks of native proteins. Compared to full-length proteins, they can be readily produced and modified by genetic engineering. Therefore, the structure and properties of protein domains can be tailored for specific applications. As one example, a segment of coiled-coil derived from a natural protein kinesin was used to crosslink a water-soluble copolymer of N-(2-hydroxypropyl)methacrylamide (HPMA).23,25 Coiled-coils are left-handed super-helical bundles of two or more right-handed R-helices that have been found ubiquitously from cell matrix proteins to transcription factors.26 The coiled-coil-cross-linked hydrogel underwent a temperature-dependent decrease in volume due to thermal unfolding and collapse of the rodlike helical protein. Hybrid hydrogels have also been assembled from an engineered immunoglobulin (Ig) domain from the giant muscle protein titin and copolymers of acrylamide.24 Cooperative unfolding of the globular Ig domain upon heating led to an expansion of the hydrogel volume. In both systems, attachment of protein cross-links to synthetic polymer backbone was accomplished through Ni(II) com-

10.1021/bm0155322 CCC: $20.00 © 2001 American Chemical Society Published on Web 06/26/2001

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(c)

Figure 1. Construction of expression vectors. The empty rectangular blocks represent the KS coiled-coil segments. (a) pET21-KS1∼3 containing one, two, and three copies of the KS segment fused in tandem. (b) Insertion of an N-terminal His-tag. (c) Sequence of H6 duplex, a linker used in part b which encodes a His-tag.

plexation involving polymer side chains and genetically inserted oligo(histidine) tags on the proteins. Our previous studies suggest that environment-sensitive conformational change of protein cross-links triggered volume transition of hybrid hydrogels, and that the responsive properties of these gels can be engineered by adjusting the sequence and structure of protein cross-links. In this paper we further examined the influence of higher-order structure and stability of coiled-coil cross-links on the properties of hybrid hydrogels. Recombinant block proteins were constructed with varying numbers of blocks based on a natural coiled-coil. The structure and solution properties of the block proteins were characterized by biophysical means and were correlated with the temperature-induced volume transition of hybrid hydrogels.

Materials and Methods Materials for Gene Cloning. The cloning vector used was pET21a (Novagen, Madison, WI) encoding a C-terminal Histag. Restriction enzymes (Bgl II, Eag I, Hpa I, AVa I, Nde I, Hind III), Klenow fragment, T4 DNA ligase, and calf intestinal alkaline phosphatase were purchased from New England Biolabs (Beverly, MA). Escherichia coli strains DH5R (Gibco BRL, Life Technologies) and BL21(DE3) (Novagen, Madison, WI) were used for plasmid amplification and protein expression, respectively. Recombinant vectors were verified by sequencing.

Construction of Expression Vectors. A previously constructed vector pET21a-CC123 was first digested with Bgl II and Eag I. The resulting 222-base-pair KS (kinesin stalk) segment was inserted into the Bgl II/Eag I sites of vector pET21a yielding pET21a-KS1. Vector pET21a-KS1 was then digested with Eag I. The resulting two cohesive ends were blunted by treatment with Klenow fragment, followed by another digestion with AVa I. This linearized vector with one blunt end and one cohesive end was ligated with the KS segment to give pET21a-KS2 (Figure 1a). By repeating the above procedure, a third copy of the KS segment was inserted into pET21a-KS2 to give pET21a-KS3 (Figure 1a). The tandemly linked KS segments were in frame. Codons in the region between segments encoded alanine residues. A His-tag was added to the N-terminus to produce proteins with His-tags at both ends (Figure 1b). First, a short chemically synthesized linker sequence encoding six-histidines (H6 duplex, Figure 1c) was inserted into the Nde I/AVa I sites of vector pET21a to give pET21-2H6. Sequential treatments of pET21a-2H6 by Hind III, Klenow fragment, and AVa I resulted in a linearized vector with a blunt end, and a cohesive AVa I end. This was then ligated with Hpa I/AVa I-digested fragments of KS1-3 to give vectors pET21aKS1∼3-2H6 (Figure 1b). Protein Expression and Purification. E. coli BL21(DE3) cells transformed with the recombinant vectors were cultured in 500 mL of LB medium containing ampicilin. After 5 h of vigorous aeration at 37 °C, protein expression was induced

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by adding isopropyl β-thiogalactoside (Sigma) to a final concentration of 0.6 mM. After inducing, incubation continued at 22 °C overnight, and cells were harvested by centrifugation. The KS block proteins were purified under native condition using immobilized metal affinity chromatography.27 Bacterial cell pellets were resuspended in Tris buffer (20 mM Tris, 500 mM NaCl, pH 6.9) containing 1 mM of phenylmethylsulfonyl fluoride (Sigma). Cell suspension was sonicated followed by centrifugation at 4 °C for 30 min. The supernatant was loaded onto a nickel affinity column (Qiagen, Santa Clarita, CA) equilibrated in Tris buffer. The column was washed sequentially with Tris buffer containing increasing concentrations of imidazole. Eluted fractions were collected and analyzed on SDS-polyacrylamide gels. The fractions containing the KS block proteins were pooled, concentrated, desalted into deionized water using prepacked Sephadex G-25 size exclusion column (Pharmacia), and lyophilized. The yields of KS1, KS2, and KS3 were 16, 22, and 30 mg per liter of culture media, respectively. Molecular weight of purified proteins was determined by matrixassisted-laser-desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Circular Dichroism. Far-ultraviolet CD spectra of the KS block proteins were recorded at 25 °C on an Aviv 62DS CD spectrometer using a 0.1 cm path length quartz cuvette. Lyophilized proteins were dissolved in 20 mM Tris plus 500 mM NaCl, pH 6.9, to prepare stock solutions of 0.3 mg/ mL. The molar concentrations of proteins (7 to 22 µM) were determined by UV spectroscopy. Scans from 200 to 250 nm were performed in 1 nm steps and 5-s data averaging at each step. The spectra obtained were averaged from three consecutive scans, background subtracted, and smoothed. Temperature-induced unfolding of proteins was monitored by recording change in ellipticity at 222 nm. The protein solutions were heated using a thermoelectric temperature control system from 15 to 95 °C at 2 °C per step. At each temperature, 4 min were given to equilibrate, and data was collected and averaged for 60 s. The buffer pH changed to 5.5 at 90 °C. To assess reversibility, samples were cooled to 25 °C after each temperature ramp, and spectrum scans were recorded and compared with those obtained before heating. Size Exclusion Chromatography and Native Gel Electrophoresis. Size exclusion chromatography was done with an A ¨ KTA fast protein liquid chromatography (FPLC) system equipped with UV and differential refractive index detectors and a Superose 12 analytical column (Pharmacia). The column was equilibrated with phosphate-buffered saline (PBS, 20 mM NaPO4 plus 150 mM NaCl, pH 7.3) containing 0.1% sodium azide and calibrated with lysozyme (14.4 kDa), carbonic anhydrase (29 kDa), bovine serum albumin (68 kDa), and IgG (150 kDa). The KS block proteins were applied to the column with an initial concentration of 0.1 mg/mL. Native gel electrophoresis was done on an 8-25% gradient polyacrylamide gel using a Phast-gel system (Pharmacia). Protein bands were visualized by silver staining. Analytical Ultracentrifugation. Sedimentation equilibrium experiments were performed with three concentrations

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(4-40 µM) of each KS block protein in PBS (A280: 0.6, 0.3, and 0.15 in a 1 cm-path length cuvette) in a Beckman Optima XL-A analytical ultracentrifuge with an An 60 Ti rotor. Samples of 110 µL volume and buffer of 125 µL volume were loaded into six-channel Epon charcoal-filled centerpieces with a 12 mm path length. Data were collected at 20 °C with rotor speeds of 15 000, 18 000, and 24 000 rpm for KS1∼3, respectively. Scans performed 4 h apart were overlaid to ensure that equilibrium was reached. Data analysis was performed using the nonlinear regression program NONLIN.28 Preparation of Metal-Chelating Copolymers. Monomers N-(2-hydroxypropyl)methacrylamide (HPMA)29 and N-[3(N′,N′-dicarboxymethyl)aminopropyl]methacrylamide (DAMA),23 were synthesized according to published procedures. Radical precipitation copolymerization in acetone was used to synthesize poly(HPMA-co-DAMA) as described previously.23 Chemical structure of the copolymer is shown in Figure 2a. Molecular weight distribution of copolymers containing different feed amount of DAMA was determined by size exclusion chromatography and content of metalchelating ligand determined by acid-base titration. The HPMA copolymer used in assembling hydrogels had a number-average molecular weight of 240 kDa, a polydispersity of 1.7, and contained 7.5 mol % of DAMA monomer units. Assembly of Hybrid Hydrogels. A solution of HPMA copolymer (200 mg/mL in 20 mM Tris, 250 mM NaCl, pH 4) was mixed with 1 mM nickel sulfate at 1:1 molar ratio of side-chain metal-chelating group to nickel ion. The KS block proteins were dissolved in the same buffer and thoroughly mixed with the copolymer-Ni(II) solution at pH 4 to prevent gelation. The solution was placed in a dialysis apparatus consisting of two layers of dialysis membrane (MWCO 1000 Da) sandwiched between three glass slides drilled with holes of 3 mm in diameter. Gelling solution of 10 µL volume was dialyzed against 20 mM Tris plus 250 mM NaCl at pH 7 for 5 h. The apparatus was then disassembled, and a gel disk (3 mm in diameter and 1 mm in thickness) was retrieved. Each gel disk contained 1 mg of the HPMA copolymer, 0.2 µmol of Ni(II), and 0.16-0.44 mg of KS block proteins. Because one block protein had two His-tags, assuming 100% cross-linking efficiency, the nominal cross-linking density was defined as 2 × (number of block protein molecules)/ (number of monomer units in the HPMA copolymer backbone) × 100% and was varied from 1.3 to 3.6 mol %. Thermal Stability of Nickel(II) Complexation in Swelling Buffer. Agarose beads bearing immobilized ligandss iminodiacetate (IDA) (Pierce) and nitrilotriacetate (NTA) (Qiagen)swere placed separately in chromatography columns and charged with Ni(II) by incubation with 200 mM nickel sulfate solution. His-tagged KS block proteins dissolved in a swelling buffer containing 20 mM Tris plus 500 mM NaCl, pH 6.9, were added to the beads and extensively washed to remove loosely bound protein. The beads were suspended in the same buffer and heated while stirring. A thermocouple was inserted into the suspension for temperature measurement. After each 10 °C increment, the beads were allowed 5 min to equilibrate and sediment, and a small

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Figure 2. (a) Chemical structure of the copolymer of HPMA and DAMA forming the backbone of hybrid hydrogels. (b) Amino acid sequence of the KS segment from the Drosophila kinesin stalk region. Single-letter abbreviation for amino acid residues is used. (c) Schematic representation of the KS block proteinssKS1, KS2, and KS3. The circles represent the terminal His-tags, through which the proteins were attached to synthetic polymer backbone by Ni(II) complexation. Striped rectangles represent the sequence with high propensity of coiled-coil formation, and empty rectangles represent the noncoiled-coil regions. Two alanine residues were placed between two adjacent KS blocks.

aliquot of the supernatant was removed and analyzed on SDS-polyacrylamide gels with Coomasie staining. The heating continued until the suspension boiled. After the suspension was cooled to room temperature, the remaining bound protein on the beads was eluted with 500 mM of EDTA and quantified by gel electrophoresis. Hydrogel Swelling and Temperature Response. Dried gel disks were rehydrated by immersion in swelling buffer and viewed under a Nikon Eclipse E800 optical microscope. Gels swelling in buffer were imaged with a CCD camera. One-dimensional size of the gels was measured from images taken at different time points. Assuming isotropic swelling, the volume swelling ratio Q was calculated as (L/L0)3, where L was the average size of swollen gels and L0 was the average size of dried gels after three separate measurements. The equilibrium volume swelling ratio Qeq was calculated as the ratio of the gel volume after 5 h in buffer vs the volume of dried gel. To test the dependence of gel swelling on temperature, equilibrated gels were placed in swelling buffer in a waterjacketed and heated glass cuvette. The heating rate was 20 °C per hour. The temperature inside the cuvette was measured directly using a digital thermocouple. Q at different temperature was determined as described above. After the gels were heated to 90 °C, they were cooled slowly to room temperature, remaining immersed in buffer for at least 12 h, and the size of the gels was measured again. Results Biosynthesis of Block Proteins. The amino acid sequence of the N-terminal segment of the Drosophila kinesin stalk

(from N340 to A410, designated as the KS segment) is shown in Figure 2b. The first 40 residues (N340 to E380) form a coiled-coil and the remaining 30 residues adopt noncoiled-coil structures forming a flexible hinge in the kinesin stalk.30,31 The DNA sequence encoding the KS segment was extracted by PCR and subcloned into a bacterial vector. Two and three copies of the KS segment were fused in tandem and were flanked by two His-tags. As illustrated in Figure 2c, the three recombinant proteins, designated as KS1, KS2, and KS3, were modular in structure containing alternating blocks of coiled-coils and noncoiled-coil chains. The terminal His-tags served both as affinity tags for protein purification and as anchors for attachment to the synthetic polymer backbone. The KS block proteins were expressed in soluble form in E. coli. A single step of Ni affinity purification resulted in >95% pure proteins detected as single bands on denaturing polyacrylamide gels stained with Coomasie Blue (Figure 3). Secondary Structure and Thermal Stability. Far-UV CD spectroscopy was used to probe the secondary structure of the KS block proteins in swelling buffer. All three proteins were predominantly R-helical as indicated by the characteristic double negative peaks at 222 and 208 nm (Figure 4a). The values of ellipticity ratio at 222 vs 208 nm were close to 1 for KS1 (0.97), KS2 (0.97), and KS3 (0.98), which suggests formation of coiled-coils.32 On the basis of molar ellipticity at 222 nm, the three proteins had the same R-helical content, consistent with the fact that they are a homologous series built with the same module. Thermal stabilities of the three proteins were measured by monitoring the change of ellipticity at 222 nm with temperature in swelling buffer (Figure 4b). KS1 underwent

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Figure 3. Analysis of protein expression by SDS-PAGE (15%). KS1: lanes 1 and 2. KS2: lanes 3 and 4. KS3: lanes 5 and 6. Total cell lysate: lanes 1, 3, and 5. Purified protein: lanes 2, 4, and 6.

a cooperative unfolding process with an onset transition temperature of 63 °C. KS2 was more stable than KS1 with an onset temperature of 75 °C. Unfolding of KS3 was biphasic with onset temperatures of approximately 63 and 75 °C. Interestingly, the first phase of transition overlapped with that of KS1, and the second phase overlapped with that of KS2 (Figure 5b, inset). After the sample was cooled to room temperature, the original ellipticity values were recovered, indicating that the block protein thermal unfolding was fully reversible. Identical results were obtained with all three block proteins in PBS (data not shown). Higher-Order Structure of KS Block Proteins. Size exclusion chromatograms (SEC) of the block proteins were recorded under nondenaturing conditions (Figure 5a). Apparent molecular weights (MW) of the proteins were calculated based on calibration of the SEC column using a series of globular proteins. KS1 eluted as a single species (peak A) with an apparent MW of a tetramer. Neither smaller nor larger oligomers were detected. Two major peaks of KS2 were found: peaks B and C having apparent MWs of a dimer and tetramer, respectively. Peak B of KS2 had the same elution time as peak A of KS1, which suggests that the KS2 dimer has a similar hydrodynamic radius as the KS1 tetramer. KS3 had one major peak (D) and one minor peak. Peak D had an apparent MW of a pentamer. It is possible that peak D was actually a tetramer whose elongated shape resulted in a larger apparent MW. The number and relative migration distance of bands observed by native gel electrophoresis (Figure 5b) corresponded to the SEC peaks. In particular, band B of KS2 migrated about the same distance as band A of KS1. Analytical ultracentrifugation (AUC) was performed to determine the oligomerization state of the KS block proteins. Sedimentation equilibrium data of all three proteins were fit with monomer-dimer, monomer-tetramer, and dimer-tetramer equilibrium models. Only the dimertetramer model fit the data reasonably well (Supporting Information). Assembly of Hybrid Hydrogels. An HPMA copolymer containing metal-chelating IDA side chains was prepared by radical polymerization. Adding Ni(II) ions to a polymer solution of neutral pH at 1:1 Ni(II)-IDA ratio resulted in a viscous gel, which dissolved upon continued mixing in buffer due to the exchange between chelated and free nickel ions. A block protein solution was added and a translucent gel formed immediately upon mixing. The stability of the KS

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block protein cross-linked gels was evaluated by incubation in swelling buffer with constant stirring for 3 h. No protein was detected in the buffer by UV spectroscopy. In contrast, when physically entrapped in a gel of HPMA copolymer cross-linked with methylenebis(acrylamide) (MBAA), over 90% of the KS block proteins diffused into the buffer within 3 h (data not shown). The gels used for the swelling experiments were assembled by mixing HPMA copolymer and the KS block proteins with Ni(II) at 1:1 IDA-Ni(II) ratio at acidic pH. Protonation of carboxylic chelating groups on the synthetic polymer side chains inhibited immediate gelation allowing better mixing of components. When dialyzed against a buffer of neutral pH, the carboxylic acid side chains were slowly deprotonated resulting in more homogeneous gels as judged by improved optical transparency. The gels contained the same amount of HPMA copolymer (1 mg) with polymer/ protein mass ratio varying from 2.3 to 6.3 and nominal crosslinking densities varying from 1.3 to 3.6 mol %. Dynamic and Equilibrium Swelling of Hydrogels. Swelling kinetics of these gels in swelling buffer is shown in Figure 6, and was typical of ionic polymeric hydrogels.33 The volume swelling ratio (Q) after 5 h was taken as the equilibrium swelling ratio (Qeq). Although swelling continued at a much slower pace for at least 24 h, accurate determination of Q became difficult because softened gels were mechanically too weak to handle and were often deformed. For the gels having the same nominal cross-linking density (1.9 mol %), there was a slight increase in Qeq following the order of KS1, KS2, and KS3 (Figure 6a). Higher nominal cross-linking density resulted in more pronounced change in Q (Figure 6b). Thermal Stability of Ni(II) Complexation. Ni(II) complexes formed by the His-tag and metal-chelating ligands such as iminodiacetate (IDA) and nitrilotriacetate (NTA) are widely used to purify His-tagged recombinant proteins.27 We used Ni(II) coordination to cross-link the KS block proteins to the synthetic polymer backbone. To examine the thermal stability of the Ni(II) complexes in swelling buffer, Ni(II)charged IDA- or NTA-agarose beads bound with Histagged block proteins were equilibrated at increasing temperatures. The amount of protein released from the beads was quantified by gel electrophoresis (Figure 7). Proteins released at lower temperature from the IDA-beads than from the NTA beads. However, with prolonged stirring at boiling temperature, less than 10% of the total amount of bound protein released from either the IDA or NTA beads, suggesting that the Ni(II) complexes are thermally stable up to 95 °C and as pH changes from 6.9 to 5.5. Temperature Dependence of Hydrogel Swelling. Gels cross-linked by the KS block proteins with a constant polymer/protein mass ratio (3.3) and therefore different nominal cross-linking densities displayed a continuous decrease in volume swelling degree at higher temperatures (Figure 8a). To evaluate the temperature response of the polymer backbone in swelling buffer, control gels were prepared using a small chemical cross-linker MBAA with nominal cross-linking density of 1 mol %.

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Figure 4. Secondary structure and thermal stability of the KS block proteins by CD spectroscopy: (a) CD spectra; (b) thermal unfolding. The arrows indicate the onset temperatures of transition (see text for details). Inset: expanded scale in a higher temperature range.

Figure 5. Higher-order structure of the KS block proteins. (a) Size exclusion chromatograms. The theoretical monomer MW and apparent MW values of individual peaks are shown in parentheses. (b) Native polyacrylamide gel electrophoresis (8-25%) with silver staining. Lane 1: KS1. Lane 2: KS2. Lane 3: KS3. SEC peaks and their corresponding bands on the electrophoresis gel are labeled with the same letter.

The slope of volume transition was steeper and the magnitude of decrease in volume was larger in gels containing KS1 and KS3 with Q90/25 of 0.32 and 0.20, respectively. The difference in volume transition between gels cross-linked with KS2 and MBAA was less pronounced. The control gels decreased in volume by approximately 40%. This reflects the temperature effect on HPMA copolymer-solvent interaction. There was no volume change of control gels between pH 6.9 and 5.5. To illustrate more clearly the contribution of the KS block proteins to hydrogel volume transition, the swelling change of MBAA cross-linked gels was subtracted from that of the protein cross-linked gels (Figure 8b). The onset temperature of volume transition was 55 ∼ 60 °C for gels cross-linked with KS1 or KS3, which corresponded reasonably well with the onset temperature of protein unfolding (63 °C). The change of volume KS2 cross-linked gels was not significantly different from the MBAA cross-linked gels.

Discussion In a previous study we assembled a hybrid hydrogel crosslinked with a recombinant coiled-coil, CC1, which was derived from the kinesin stalk region.23 CC1 contained extended R-helical coiled-coil structure spanning approximately 250 amino acid residues with a total length estimated to be 25-30 nm.34 A sharp 90% drop in hydrogel volume occurred upon heating, and the mid-transition temperature lay in the vicinity of the Tm of CC1 (39 °C).23 We hypothesized that the underlining mechanism for this gel volume transition could be the large decrease in hydrodynamic volume of the rodlike protein cross-link upon unfolding.25 In fact, the theoretical value of the radius of gyration (RG) for a 150-amino acid protein is 65 Å if it is R-helical, 43 Å if it is a random coil, and 12 Å if it is a compact sphere.35 To further test our hypothesis, we constructed three block proteins with increasing numbers of blocks which consisted of interspersed coiled-coil and noncoiled-coil

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Figure 6. Swelling kinetics of a series of hybrid hydrogels containing KS block proteins with the same (a) and varying nominal cross-linking density (b). The equilibrium swelling degree Qeq was inversely proportional to the nominal cross-linking density.

Figure 7. Thermal stability of Ni(II)-His-tag complexation with iminodiacetate (IDA) and nitrilotriacetate (NTA). The amounts of protein released from ligands at increasing temperatures were expressed as intensities of gel electrophoresis bands and normalized against the total amount of protein (last column “EDTA”). Less than 10% of the total amount of bound protein was released during heating.

modules. It was expected that (1) the hydrogel volume transition would be determined by the thermal stability of the coiled-coil block proteins and (2) the magnitude of volume transition would be directly related to the hydrodynamic radius of the block proteins. A Model of Block Protein Structure. We found that, instead of adopting extended linear structures, the KS block proteins displayed more complicated higher-order structures and an oligomerization state, as well as thermal stabilities. Unlike most synthetic block copolymers such as polyurethane, these macromolecular complexes were stabilized through highly specific interactions between the coiled-coil domains. On the basis of analysis of the KS block protein higher-order structure and temperature stability, we propose a hypothetical structural model for the block proteins (Figure 9), which is described as follows: (I) In equilibrium sedimentation experiments self-association of the KS block proteins was best described by a dimertetramer equilibrium. SEC and native gel electrophoresis suggested that KS2 dimer had a hydrodynamic radius similar to that of KS1 tetramer. Therefore, we speculate that KS2 dimer had a globular structure of similar dimensions to the

KS1 tetramer, rather than an extended linear conformation. To account for these observations, we think that the most likely structure is an antiparallel intramolecular arrangement of coiled coils (Figure 9). (II) Because the KS1 mutant cross-linked through a cysteine at the N-terminus was a stable dimer (not unfolding >90 °C, data not shown), the unfolding process was likely initiated at the N-terminus (highlighted in gray in Figure 9). (III) KS3 displayed biphasic thermal melting with two transition temperatures corresponding to those of KS1 and KS2, respectively (Figure 4b). Therefore, we propose a structure of KS3 consisting of a KS1 module on top of a KS2 module (Figure 9). Independent unfolding of the two modules initiated at the N-termini would explain the biphasic unfolding of KS3. Swelling of Hydrogels. The hybrid hydrogels reported here consist of synthetic hydrophilic polymer backbones cross-linked by block protein chains with complicated threedimensional structures. To keep the polymer backbone contribution to swelling constant, the same polymer mass was used in gel assembly. The only variables were the type and mass of the block proteins as well as the nominal crosslinking density assuming 100% cross-linking efficiency by the block proteins. Gels with the same nominal cross-linking density showed only slight differences in swelling degree despite being crosslinked with different KS block proteins (Figure 6a). The length of the fully extended chains of the KS block proteins differs considerably, which should have resulted in gels with large difference in volume swelling degree. That the difference in gel volume was much smaller suggested that the actual size of the block proteins was also smaller, probably because of intramolecular folding as hypothesized in our structural model (Figure 9). Different polymer/protein molar ratios resulted in considerable differences in gel swelling degree (Figure 6b) indicating that the nominal cross-linking density and three-dimensional structure of the block proteins are important factors influencing gel swelling.

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Figure 8. Temperature dependence of swelling of hybrid hydrogels cross-linked by the block proteins and methylenebis(acrylamide) (MBAA). Q/Qeq is the ratio of gel volume at elevated temperature to the equilibrium swelling degree at room temperature: (a) change in Q/Qeq with temperature; (b) change after subtracting the contribution of MBAA-cross-linked polymer backbone. The arrows point to the onset time of volume transition of gels cross-linked by KS1 and KS3.

Figure 9. Hypothetical model for the higher-order structure and thermal unfolding of the KS block proteins. Cylinders depict helical regions, thick lines are noncoiled-coil sequences, and thin lines are unfolded chains. The amino termini of the protein blocks are highlighted in gray. The dominant species are labeled tentatively based on SEC peaks and native gel bands (see Figure 5).

Volume Transition of Hydrogels. Elevating temperature resulted in a continuous decrease in volume of hydrogels cross-linked with the block proteins (Figure 8). Using an HPMA copolymer cross-linked covalently by a small molecule MBAA for comparison, pronounced volume transition was only observed in gels cross-linked by KS1 and KS3, but not KS2. The transitions started at approximately 55 ∼ 60 °C (Figure 8) which was close to the onset temperature of unfolding KS1 and KS3 (63 °C, Figure 4b). Although KS2 had the medium chain length among the three proteins, gels cross-linked by KS2 displayed the smallest volume response (Figure 8). One possible explanation is that KS2 had a smaller RGsprobably due to a globular folded

structure (Figure 9)sand hence a smaller change in hydrodynamic radius between folded and unfolded states. In addition, if our model is correct, the His-tags of KS2 were clustered on one side of the complex making KS2 a less efficient cross-link than KS1 and KS3, which might also result in a less pronounced volume transition of KS2-cross-linked gels. A complete recovery of the initial gel volume was not achieved after cooling. This was not unexpected because conformational transition of large molecules such as protein36,37 and DNA38 is known to be impeded in a hydrogel environment due to steric hindrance posed by the surrounding hydrophilic polymer chains. Furthermore, tethered ends

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restricting degrees of freedom of the protein chains made it difficult for them to refold. Conclusion A series of recombinant block proteins containing different numbers of coiled-coil domains arranged in tandem were synthesized. The molecular architecture of these proteins was reminiscent of traditional synthetic block copolymers but more complicated and ordered in three-dimensions. Hybrid hydrogels of synthetic polymer physically cross-linked with the block proteins were assembled in aqueous condition. Temperature-induced volume response of hydrogels reflected the thermal stability of the block proteins in solution. The hydrogel responsiveness is determined by the higher-order structure of the block proteins resulting from inter- and intramolecular interactions among coiled-coil domains. These results demonstrated the feasibility of tailoring the stimuliresponsive behavior of hydrogels by engineering genetically the structure and properties of the protein cross-links. With further elucidation of structure-property relationship, these responsive hybrid hydrogels can be used for a variety of biomedical applications. Acknowledgment. This work was supported in part by an NSF grant (BES9807287). We thank Drs. Lisa Joss and David Myszka for assistance in analytical ultracentrifugation analysis. Supporting Information Available. Figures showing the sedimentation equilibrium results for the block proteins. This material is available free of charge via the Internet at http:// pubs.acs.org.

Wang et al. (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35)

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