Artificial Protein Block Polymer Libraries Bearing Two SADs: Effects of

Oct 26, 2011 - ‡Skirball Institute Image Core Facility, New York University Medical Center, New ... domains (SADs) derived from elastin (E) and the ...
0 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/Biomac

Artificial Protein Block Polymer Libraries Bearing Two SADs: Effects of Elastin Domain Repeats Min Dai,† Jennifer Haghpanah,† Navjot Singh,† Eric W. Roth,‡ Alice Liang,‡ Raymond S. Tu,§ and Jin Kim Montclare*,†,∥ †

Department of Chemical and Biological Sciences, Polytechnic Institute of NYU, Brooklyn, New York 11201, United States Skirball Institute Image Core Facility, New York University Medical Center, New York, New York 10016, United States § Department of Chemical Engineering, City College of New York, New York, New York 10031, United States ∥ Department of Biochemistry, SUNY Downstate Medical Center, Brooklyn, New York 11203, United States ‡

S Supporting Information *

ABSTRACT: We have generated protein block polymer EnC and CEn libraries composed of two different self-assembling domains (SADs) derived from elastin (E) and the cartilage oligomeric matrix protein coiled-coil (C). As the E domain is shortened, the polymers exhibit an increase in inverse transition temperature (Tt); however, the range of temperature change differs dramatically between the EnC and CEn library. Whereas all polymers assemble into nanoparticles, the bulk mechanical properties of the E nC are very different from CEn. The EnC members demonstrate viscolelastic behavior under ambient conditions and assemble into elastic soft gels above their Tt values. By contrast, the CEn members are predominantly viscous at all temperatures. All library members demonstrate binding to curcumin. The differential thermoresponsive behaviors of the E nC and CEn libraries in addition to their small molecule recognition abilities make them suitable for potential use in tissue engineering and drug delivery.

■

elastin (n = 5). To expand the thermoresponsive range of these materials, a library of EnC and CEn diblock polymers was constructed in which the E domain was systematically truncated, where n ranged from 4 to 1 repeats (Figure 1). We hypothesized that as the E domain is shortened, the Tt would increase based on

INTRODUCTION The fabrication of stimuli-responsive, multifunctional nanomaterials that can self-assemble into defined structures bears tremendous potential in drug delivery and regenerative medicine.1−4 Whereas there has been remarkable progress in synthetic selfassembling systems, nature provides a wealth of highly ordered structures with defined features from the nano- to the mesoscale level. In fact, a large fraction of the structures in nature are composed of proteins. Proteins not only provide a diversity of chemical functionality as the building blocks are composed of 20 amino acids but also present critical 3D structures that provide order on the nano- to meso-length scales. Inspired by nature and fueled by recent advances in molecular and synthetic biology,5,6 we7,8 and others9−22 have generated “smart” protein polymers capable of self-assembling and responding to external stimuli. Recently, we engineered three protein block polymers 7,8 −EC, CE and ECE−composed of two distinct self-assembling domains (SADs) derived from elastin (E) 23,24 and cartilage oligomeric matrix protein coiled-coil (C). 25,26 Characterization of the proteins revealed that the orientation and number of blocks influenced the overall secondary structure, stability, supramolecular assembly, and small-molecule binding ability. Whereas the protein polymers exhibited interesting physicochemical properties, their thermoresponsive behaviors were limited; all three proteins possessed inverse temperature transitions (Tt values) at room temperature or below. The E domain length for the three constructs possessed a five repeat © 2011 American Chemical Society

Figure 1. (a) Illustration of the EnC and CEn protein block polymers and their sequences where n represents the number of E repeats. (b) 12% SDS-PAGE of purified protein polymers. Received: August 2, 2011 Revised: September 22, 2011 Published: October 26, 2011 4240

dx.doi.org/10.1021/bm201083d | Biomacromolecules 2011, 12, 4240−4246

Biomacromolecules

Article

previous literature on elastin-based polypeptides.22 Whereas an increase in Tt was observed upon decreasing the E domain length as expected, the orientation of the diblocks affected the secondary structure, supramolecular assembly, mechanical properties, and small molecule recognition.

■

Table 1. Protein Polymer Yields and Molecular Weights

MATERIALS AND METHODS

General. BamHI, SacI, SalI, HindIII and dNTPs were obtained from Roche Applied Science, whereas PicoMaxx high fidelity was purchased from Agilent technologies. Yeast extract and curcumin were obtained from Acros Organics. Tryptic soy agar was acquired from MP Biomedicals. Ampicillin, isopropyl β-D-1-thiogalactopyranoside (IPTG), imidazole, sodium monobasic phosphate, sodium dibasic phosphate, sodium hydroxide, sodium chloride, sucrose, tryptone, and urea were obtained from Fisher Scientific. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), magnesium sulfate, and nickel chloride were purchased from Sigma Aldrich. Ethylenediaminetetraacetic acid (EDTA), hydrochloric acid was acquired from VWR. HPLC grade methanol was obtained from Ricca Chemical Company. Sinapinic acid was purchased from Thermo Scientific. Protein standards used for MALDI MS were acquired from New England Biolabs. Construction of DNA Libraries. The following primers were used to amplify the library of E domain length from pUC19ELP pentamer (gift from D. Tirrell) plasmid: BamHI:5′-ggaggccGGATCCaagccgattgcggctagcgcggtgccgg-3′, SacI:5′-gccccGAGCTCcgatccctcgagcggcaccccgac-3′, SalI:5′-ggaggccGTCGACaagccgattgcggctagcgcggtgccgg-3′, and HindIIII: 5′-ggccccAAGCTTcgcaccggtacccgatccctcgagcggcaccccgac-3′. Fragments of E of 150, 200, 300, and 350 bps were amplified by a combination of increasing the plasmid DNA to 300 ng/μL, decreasing the number of cycles and decreasing the annealing temperature. The inserts were gel-purified and restricted with appropriate restriction enzymes. E inserts were cloned into the PCR-assembled pQE30/C between restriction sites BamHI and SacI to generate pQE30/EnC or SalI and HindIII yielding pQE30/CEn. The clones were verified by forward and reverse DNA sequencing (MWG operon). Expression and Purification. The resulting plasmids were transformed into XL1-blue cells and expressed according to previously published protocols.7,8 Transformed library members were cultured in 1 L LB media bearing 0.57 mM ampicillin at 37 °C until the optical density at 600 nm (OD600) reached ∼0.6. IPTG was added to 0.52 mM final concentration to induce overexpression for overnight. Cells were harvested and resuspended to 7 mL of lysis buffer (buffer A: 6 M urea, 50 mM Na2HPO4, 20 mM imidazole, pH 8.0), the cell suspensions were stored at −80 °C. The suspension was thawed at 4 °C, osmotically shocked27 by incubation with 40 mL of sucrose buffer (50 mM HEPES, 20% sucrose, 1 mM EDTA pH 7.9), and pelleted. Supernatants were discarded, and pellets were resuspended in 25 mL of 5 mM MgSO4, incubated on ice for 10 min, and harvested. Cell pellets were resuspended in 25 mL of buffer A and subjected to lysis via French press (Thermo Scientific). The proteins in the lysate was purified via FPLC using a HiTrap IMAC FF column (5 mL volume) that was charged with NiCl2 and equilibrated with lysis buffer using an ATKA purifier system (GE Life Sciences). Protein was eluted by adjusting percentage of buffer B (same components as buffer A but with 200 mM Imidazole) with 50 mL of 1%, 30 mL of 5%, and 50 mL of 100% at 5 mL/min flow rate. The elutions bearing purified protein were collected and dialyzed against 10 mM phosphate buffer pH 8.0 at 4 °C. Purity was confirmed by SDS-PAGE and ImageQuant-TL 1D gel analysis program prior to dialysis (GE Life Sciences). Concentrations of protein were determined via micro-BCA analysis (Thermo Scientific) by using SpectraMax M2 (Molecular Devices). Molecular weights were confirmed via matrix-assisted laser desorption ionizationtime-of-flight mass spectrometry (MALDI-TOF MS) on a Bruker Omniflex (Table 1). Circular Dichroism. Wavelength-dependent circular dichroism (CD) spectra were collected on a Jasco J-815 CD spectrometer equipped with a PTC-423S single position Peltier temperature control system and counter-cooled with an Isotemp 3016S (Fisher Scientific)

protein

purified yields (mg L )

calculated molecular weight (Da)

E1C E2C E3C E4C CE1 CE2 CE3 CE4

5.9 11.5 6.7 10.1 3.0 4.3 3.9 7.7

13865 15960 18072 20151 13942 16037 18132 20141

−1

observed molecular weight from MALDI (Da) 13871 15884 18037 20034 13959 16089 18191 20143

water bath. Samples were loaded in a Hellma 218 quartz cuvette (500 μL, 1 mm path length). A far-UV temperature-dependent wavelength scan from 185 to 260 nm as a function of temperature was completed for EnC and CEn library members at 10 μM in 10 mM phosphate buffer pH 8.0 at scan rate of 50 nm/min at 1 °C/min temperature increasing rate. All scans were performed three times for accumulation. CD data was converted into mean residue molar ellipticity ([θ]mrw) via an equation [θ]mrw = θ·MRW/(10·C·l), where θ is the data obtained in mdeg, MRW is mean residue weight, C is concentration in milligrams per milliliter, and l is path length in centimeters.28 Fitting and calculation of protein secondary structure was processed with CDSSTR methods.29−32 UV/vis Spectrometry. The inverse temperature transition (Tt) was determined using UV−vis instrument Cary-50 (Varian) with TC125 temperature controller (Quantum Northwest) by observing the change in turbidity at 320 nm from 15 to 80 °C at 1 °C/min temperature increasing rate. Protein samples were prepared in 0.2 mg/ mL in 10 mM phosphate buffer, 500 mM NaCl, pH 8. Samples were loaded in type 21 quartz cuvette with 10 mm path length (Buck Science). Scans were performed on at least two different protein sample preps to calculate the average Tt. Tt was determined at the inflection point of the absorbance curve.33 Transmission Electron Microscopy. Transmission electron microscopy (TEM) was used to identify the potential nanometersized structures that resulted from self-assembly at 4 °C. Samples were prepared in water at 0.2 mg/mL concentrations in 10 mM phosphate, 500 mM NaCl pH 8.0, and gently vortexed. The samples were applied on a carbon-coated 400 mesh Cu/Rh grids and negatively stained with 1% uranyl acetate. The images of the samples were collected on a Phillips CM12 Tungsten Emission TEM at 120 kV. The particle area and size were measured using ImageJ.34 Sizes of all particles were determined from at least >25 particles from two separately purified protein samples. Microrheology. Lypohilized protein samples were resuspended in 10 mM phosphate buffer, 500 mM NaCl, pH 8.0 at a final concentration of 15 mg/mL. We added 2 μL of 2% (wt %) of fluorescent amidated polystyrene beads (1.0 μm) to 10 μL of the resuspended protein sample. Epifluorescence was monitored on an inverted Nikon microscope. A Linkam LST120 peltier cell was used to control temperature. All samples were analyzed in replicate both below and above their Tt values (E1C = 45 °C, E2C = 42 °C, E3C = 40 °C, E4C = 30 °C, CE1 = 64 °C, CE2 = 53 °C, CE3 = 37 °C, and CE4 = 33 °C). Samples were equilibrated at their appropriate temperatures for ∼4 min on the temperature stage prior to their run. Videos were recorded in triplicate from various locations on the slide with a QiCam (640 × 480 pixels at 30 and 60 fps) and converted to 8-bit tiff file for IDL analysis. Particle trajectories were obtained from three videos with IDL image software analysis, and the dynamic moduli were determined using the Stokes− Einstein relation.35−40 Fluorescence. Protein stock samples were prepared at 6 μM concentration in 10 mM phosphate buffer, pH 8. Curcumin was freshly dissolved in HPLC grade methanol, followed by dilution into 10 mM phosphate buffer pH 8 to give stock standards ranging from 1.5 to 54 μM. Proteins were loaded in a Costar 96-well black plate (Corning Life Science); subsequently, curcumin standards were added using Biomek 4241

dx.doi.org/10.1021/bm201083d | Biomacromolecules 2011, 12, 4240−4246

Biomacromolecules

Article

NXP Laboratory Automation Workstation (Beckman Coulter) to start all binding reactions at the same time. Final protein polymer concentration was 4 μM, and curcumin concentrations ranged from 0.5 to 18 μM.8 For each experiment, three replicates of curcumin standards and two replicates of protein with curcumin were prepared. The average dissociation constant (Kd) was calculated from two separate batches of proteins from two different expressions. Fluorescence was recorded after 2 h of incubation by using SpectraMax M2 (Molecular Devices) with 420 nm excitation, 495 nm emission, and 30 readings per well. Fitting and determination of Kd were carried with Sigmaplot using one site saturation without nonspecific binding equation: Fb = Fs[L]/(Kd + [L]) in which Fb is the fluorescence signal of bound ligand, [L] is the ligand concentration in micromolar, and Fs is the plateau signal at saturation.25

Figure 2. Characterization of inverse thermal transitions. (a) Turbidity profiles for E1C (circle), E2C (diamond), E3C (triangle), and E4C (square) of the EnC library. (b) Turbidity profiles for CE1 (circle), CE2 (diamond), CE3 (triangle), and CE4 (square) of the CEn library.

■

CEn counterparts possessed nearly identical composition and molecular weights. Temperature-Dependent Secondary Structure. To determine the effects of E domain truncation on the overall secondary structure of the EnC and CEn libraries, far-UV wavelength scans were performed as a function of temperature (Figure 3). In our previous work, we demonstrated that the diblocks EC and CE were structurally different even though they were nearly identical in composition.7 As expected, the EnC and CEn diblocks differed in secondary structure and exhibited different temperature-dependent conformational changes (Figure 3, Supporting Information). The E4C was predominantly unordered or random-like at 4 °C, and upon increasing temperature, it assumed more β-conformation. A similar trend was observed for E3C, in which the initial structure was more random-like and the final structure was less β-like relative to E4C. Interestingly a shift in the trend was observed for E2C; at 4 °C, it was helical and random-like, and at elevated temperature it was predominantly β-like in conformation. The E1C exhibited a helical signature and a final structure that was mostly β-structured with almost an equal amount of random conformation. For the CEn library, all proteins exhibited a significant random-like conformation at 4 °C and became more β-like with a substantial unordered population. As the E domain was shortened, the starting structure was less random with more helical contribution, and the final structure at elevated temperatures was more β-like yet maintained a predominantly unordered conformation for the CEn series. Essentially, the decrease in E length led to a reduction in β-conformation and increase in helical contribution from the C domain for both series at low temperatures. Supramolecular Particle Assemblies. Because previous studies showed that such protein block polymers could assemble into supramolecular structures, the library members were visualized via TEM to assess the assemblies formed. All proteins revealed the formation of discrete nanometer-sized particles confirming the supramolecular assemblies at 0.2 mg/mL concentrations (Figure 4, Supporting Information). In the case of the EnC library members, the particles ranged from 26.4 ± 3.8 to 28.5 ± 3.1 nm, whereas for CEn library members the particles ranged from 26.0 ± 3.0 to 30.4 ± 6.1 nm (Table 2). The truncation of the E domain for both libraries did not substantially influence the size of the particles because they all were within the same size range. Temperature-Dependent Mechanical Properties. To determine the mechanical properties of the protein polymer library, microrheology was performed at 12.5 mg/mL protein concentrations. In the case of the EnC library at 22 °C, all proteins exhibited viscoelastic behavior with a crossover of

RESULTS Biosynthesis of Protein Libraries. To create the EnC and CEn libraries in which the E domain was systematically truncated, we PCR amplified the E gene using primers bearing BamHI and SacI resitriction sites or SalI and HindIII restriction sites. The E fragments ranging from 150 to 350 bps were cloned into the parent plasmid pQE30/C (bearing the C domain) to produce pQE30/EnC or pQE30/CEn.7 This produced library members in which one to four repeats of the E domain was expressed (Figure 1). The protein polymer library members were overexpressed, purified, and characterized. After affinity purification, 3.0−11.3 mg was recovered for each protein polymer (Table 1). Whereas SDS-PAGE analysis demonstrated slightly higher molecular weights for EnC and CEn (Figure 1B), the exact molar masses were confirmed by MALDI-TOF MS (Table 1). Inverse Transition Temperature. We hypothesized that shortening the E domain length would cause an increase in the Tt and overall thermoresponsive behavior of the protein block polymers. To test this, we monitored the UV/vis absorbance of the EnC and CEn library members as a function of temperature. All protein polymers exhibited incrementally elevated Tt values upon truncation of E at 0.2 mg/mL concentrations (Table 2, Figure 2). Table 2. Supramolecular Assembly and Small Molecule Binding of Protein Library protein E1C E2C E3C E4C CE1 CE2 CE3 CE4

Tt (°C)a 38.0 35.3 31.0 28.5 59.0 45.4 35.0 27.0

± ± ± ± ± ± ± ±

0.0 5.5 0.0 2.1 5.9 2.6 2.9 2.0

particle size (nm)b 27.9 26.4 28.5 27.6 26.0 30.4 26.0 28.2

± ± ± ± ± ± ± ±

4.5 3.8 3.1 4.2 3.0 6.1 4.1 5.6

Kd (μM)c 4.7 4.7 14.3 12.8 7.0 11.1 17.0 12.5

± ± ± ± ± ± ± ±

0.7 0.9 2.5 4.4 0.9 1.1 3.0 3.7

a

Values are obtained from an average of at least three trials of two independent protein preps. bSizes determined from TEM using ImageJ. cCalculated values are determined and an average of at least two trials of two independent protein preps.

Whereas E4C and CE4 illustrated similar Tt values of 28.5 and 27.0 °C, respectively, the rest of the library members revealed different values. The EnC library presented a narrow range of Tt values with a maximal difference of 9.5 °C when comparing E4C to E1C. By contrast, the CEn library presented a more broad temperature range with a 32 °C maximal difference. The degree by which the temperature changed was dependent on the orientation of the diblocks as the EnC and 4242

dx.doi.org/10.1021/bm201083d | Biomacromolecules 2011, 12, 4240−4246

Biomacromolecules

Article

Figure 3. Secondary structure analysis of EnC and CEn protein block polymers. Temperature-dependent wavelength scans of (a) E 1C, (b) E2C, (c) E3C, (d) E4C, (e) CE1, (f) CE2, (g) CE3, and (h) CE4.

values. Whereas the binding abilities of the E nC and CEn libraries were similar for constructs bearing the longer E repeats, both E2C and E1C showed better binding than their CEn couterparts.

■

DISCUSSION The orientation of the SADs influences the overall physicochemical properties across the EnC and CEn library members.7,8 This is most evidenced by the effects of E length for each library set on Tt, secondary structure, mechanical properties, and binding abilities to some extent. Whereas there are observable trends in overall physicochemical properties that are dependent on the E length, the trends are highly reliant on the EnC or CEn library subset. Later we explore the interplay between the different properties and assess such relationships. Interplay Between Inverse Temperature Transition and Conformation. Because the temperature-dependent phase separation is predominantly dictated by the E domain, the observed trend in Tt for both the EnC and CEn libraries could be explained by the attraction and ordering between E domains, decreasing the solvation energy (Figure 2, Table 2).52,53 This is further partially supported by the temperaturedependent CD experiments. Whereas the wavelength scans were performed in the absence of salt to prevent phase separation and light scattering during the runs (Figure 3), the conformational changes can be meaningfully interpreted with respect to the Tt. As the E domain is lengthened (i.e., E4C and CE4), localized segregation due to formation of more distinct β-like conformation at elevated temperatures could be readily induced requiring less energy or heat, leading to a lower Tt. Surprisingly, the EnC library showed a narrow difference in temperature, whereas the CEn library exhibited a more broad temperature range (Figure 2, Table 2). While orientation dependence on expression level was observed for elastin-like polypeptide fusions to other functional proteins by Chilkoti and coworkers,54 here we identified an orientation dependence on the Tt. The EnC library members illustrated more β- and α-like, ordered initial structure for the shorter repeats (n = 1, 2) in which the Tt differences are small relative to their CE n counterparts, whereas the higher repeat constructs (n = 3, 4) exhibited more similar Tt values (Supporting Information). Because elastins undergo phase separation through the ordering of random to β-conformation, 22,53,55,56 the temperature required for the already ordered EnC library members would

Figure 4. Supramolecular particle formation of (a) E1C, (b) E2C, (c) E3C, (d) E4C, (e) CE1, (f) CE2, (g) CE3, and (h) CE4. Scale bar represents 200 nm.

the G′ and G″ occurring at high frequencies (Figure 5a−d). To assess whether mechanical behavior of the protein block polymers could be affected by temperature, we performed microrheology above the Tt values for each library member (Figure 5). Remarkably, all E nC proteins demonstrated enhanced mechanical properties in which the samples revealed predominantly elastic character above the Tt. For the CEn library, all samples were viscous in contrast with the EnC (Figure 5e−h). In addition, the viscous nature of CEn was minimally altered upon incubation at temperatures above the Tt. The orientation of the diblocks significantly impacted the bulk mechanical properties of the materials. In particular, the presentation of the E domain at the N-terminus demonstrated a more pronounced effect on the mechanical behavior, leading to temperature-dependent gel formation. Small Molecule Binding Ability. Because the protein block polymer libraries possess a C domain capable of recognizing a range of small hydrophobic molecules, we monitored the binding of EnC and CEn to curcumin. We selected curcumin because it bears unique pharmacological activity,41,42 including antitumor,43−45 antiamyloid,46,47 and antihypertrophic48−51 properties, and the binding abilities can be readily monitored via fluorescence. All protein block polymers were able to bind to curcumin with micromolar affinity (Table 2). For the E nC library, the Kd values ranged from 14.2 to 4.7 μM in which E4C and E3C both possessed similarly high values (Table 2). The remaining two members bearing the shorter E domain exhibited enhanced binding of equal magnitude. The CE n library demonstrated Kd values ranging from 17.0 to 7.0 μM in which the CE3 and CE1 possessed the highest and lowest values, respectively (Table 2). The other two protein diblocks revealed intermediate Kd 4243

dx.doi.org/10.1021/bm201083d | Biomacromolecules 2011, 12, 4240−4246

Biomacromolecules

Article

Figure 5. Microrheology data of EnC and CEn library members at 22 °C (gray) and above their respective Tt values (black). Plots of G′ (closed circles) and G″ (open circles) as a function of frequency for (a) E1C (45 °C), (b) E2C (42 °C), (c) E3C (40 °C), (d) E4C (30 °C), (e) CE1 (64 °C), (f) CE2 (53 °C), (g) CE3 (37 °C), and (h) CE4 (33 °C). The numbers in parentheses denote the temperatures above Tt for each protein.

be minimal. By contrast the CEn series were predominantly unordered even at elevated temperatures. Therefore, more heat would be required to cause structure/ordering to induce phase separation especially for the CE1 and CE2 constructs relative to their EnC counterparts. Impact of Conformation on Mechanical Properties. The EnC and CEn libraries demonstrated very different bulk rheological properties, affirming the block orientation dependence on supramolecular assemblies (Figure 5).8 The ability of the EnC library to assume more elastic character could be attributed to the more β-like conformation observed at elevated temperatures when compared with the CEn library. Because the driving force for phase transition in elastin-like polypeptides has been attributed to the ordering or formation of a β-conformation, leading to the association and aggregation,52,53 the formation of conformational aggregates enhances the mechanical properties as more ordered networks are formed. Through the interactions of the E domain in the β-structured state, the EnC members could form loose networks leading to the observed viscoelastic behavior (Figure 5a−d). Upon increase in the temperature above their respective Tt values, each EnC polymer possesses more β-like conformation, which could strengthen the networks, leading to the elastic, gel-like character observed. Other rationally designed β-conformation peptides have been observed to form elastic networks though intramolecular folding and intermolecular self-assembly of the strands.38,57,58 The enhanced mechanical properties are attributed to defect-induced branching and entanglement.57 Whereas the EnC constructs are different from the β-hairpin peptides, the branching and entanglement due to β-conformation offers a potential mechanism that can be further explored. Influence of Conformation and Small Molecule Binding. A general trend was observed in which as the E domain was shortened below three repeats for both E nC and CEn, the Kd values improved (Table 2). At long E domains, the

more localized aggregation may limit the accessibility of the C domain in the EnC and CEn block polymers, leading to poor binding, whereas the short E domain does not occlude the C domain for curcumin recognition. In addition, the observed trend could be attributed to the dominance of the C domain within the block polymers, as observed by the gain of helical conformation or the loss of unordered component for the starting structures (at low temperatures) upon E truncation to 1 and 2 repeats for EnC (Figure 3a,b) and CEn (Figure 3e,f), respectively. The fact that the Kd values were strongest for both E1C and E2C relative to their corresponding CEn counterparts may be due to the stronger helical component, indicative of a folded C domain poised to recognize curcumin.

■

CONCLUSIONS Our studies demonstrate that the decreasing the E domain size can indeed modulate the thermoresponsive properties of the protein block polymers and obtain a range of Tt values, especially those above biological conditions of 37° C for further applications in biomedical research. For the EnC library, whereas the Tt range is narrow, the polymers exhibit viscoelastic character that upon heating above their respective Tt values, assemble into gel. These diblocks may be suitable for applications in tissue engineering because they are fluid-like at room temperature and upon elevated temperatures can assemble into soft gels.59−62 Such types of stimuli-responsive materials can be potentially employed as injectable scaffold. In the case of the CEn library, all protein polymers are predominantly viscous at room temperature with two members exhibiting viscoelastic character above the Tt. Whereas these diblocks may not be suitable as scaffolds for tissue, they may be useful for applications in drug delivery because they remain as nanoparticles within the solution and can indeed bind curcumin or potentially other therapeutic small molecules.8,26 4244

dx.doi.org/10.1021/bm201083d | Biomacromolecules 2011, 12, 4240−4246

Biomacromolecules

Article

Biomaterials; Cheng, H. N., Gross, R. A., Eds.; Oxford University Press: Oxford, U.K., 2008. (25) Guo, Y.; Bozic, D.; Malashkevich, V. N.; Kammerer, R. a.; Schulthess, T.; Engel, J. EMBO J. 1998, 17, 5265. (26) Gunasekar, S. K.; Asnani, M.; Limbad, C.; Haghpanah, J. S.; Hom, W.; Barra, H.; Nanda, S.; Lu, M.; Montclare, J. K. Biochemistry 2009, 48, 8559. (27) Magnusdottir, A.; Johansson, I.; Dahlgren, L.-G.; Nordlund, P.; Berglund, H. Nat. Methods 2009, 6, 477. (28) Lee, D. L.; Mant, C. T.; Hodges, R. S. J. Biol. Chem. 2003, 278, 22918. (29) Sreerama, N.; Venyaminov, S. Y.; Woody, R. W. Anal. Biochem. 2000, 287, 243. (30) Whitmore, L.; Wallace, B. A. Biopolymers 2008, 89, 392. (31) Whitmore, L.; Wallace, B. A. Nucleic Acids Res. 2004, 32, W668. (32) Sreerama, N.; Woody, R. W. Anal. Biochem. 2000, 287, 252. (33) Nuhn, H.; Klok, H.-A. Biomacromolecules 2008, 9, 2755. (34) Abramoff, M. D. M.; Paulo, J.; Ram, S. J. Biophotonics Int. 2004, 11, 6. (35) Veerman, C.; Rajagopal, K.; Palla, C. S.; Pochan, D. J.; Schneider, J. P.; Furst, E. M. Macromolecules 2006, 39, 6608. (36) Gardel, M. L.; Valentine, M. T.; Weitz, D. A. Microscale Diagnostic Techniques; Breuer, K. S., Ed.; Springer: Berlin, 2005. (37) Breedveld, V.; Pine, D. J. J. Mater. Sci. 2003, 38, 4461. (38) Schneider, J. P.; Pochan, D. J.; Ozbas, B.; Rajagopal, K.; Pakstis, L.; Kretsinger, J. J. Am. Chem. Soc. 2002, 124, 15030. (39) Mason, T. G.; Ganesan, K.; van Zanten, J. H.; Wirtz, D.; Kuo, S. C. Phys. Rev. Lett. 1997, 79, 3282. (40) Mason, T. G.; Weitz, D. A. Phys. Rev. Lett. 1995, 74, 1250. (41) Shishodia, S.; Sethi, G.; Aggarwal, B. B. Ann. N.Y. Acad. Sci. 2005, 1056, 206. (42) Shehzad, A.; Lee, Y. S. Drugs Future 2010, 35, 113. (43) Mehta, K.; Pantazis, P.; McQueen, T.; Agarwal, B. Anticancer Drugs 1997, 8, 471. (44) Cheng, A. L.; Hsu, C. H.; Lin, J. K.; Hsu, M. M.; Ho, Y. F.; Shen, T. S.; Ko, J. Y.; Lin, J. T.; Lin, B. R.; Wu, M. S.; Yu, H. S.; Jee, S. H.; Chen, G. S.; Chen, T. M.; Chen, C. A.; Lai, M. K.; Pu, Y. S.; Pan, M. H.; Wang, Y. J.; Tsai, C. C.; Hsieh, C. Y. Anticancer Res. 2001, 21, 2895. (45) Bisht, S.; Feldmann, G.; Soni, S.; Ravi, R.; Karikar, C.; Maitra, A. J. Nanobiotechnol. 2007, 5, 3. (46) DaSilva, K. A.; Shaw, J. E.; McLaurin, J. Exp. Neurol. 2010, 223, 311. (47) Darvesh, A. S.; Carroll, R. T.; Bishayee, A.; Geldenhuys, W. J.; Van der Schyf, C. J. Expert Rev. Neurother. 2010, 10, 729. (48) Suzuki, H.; Morimoto, T.; Sunagawa, Y.; Kawamura, T.; Wada, H.; Uemura, K.; Simazu, A.; Kimura, T.; Fujita, M.; Hasegawa, K. J. Card. Failure 2009, 15, S178. (49) Sunagawa, Y.; Morimoto, T.; Kawamura, T.; Takaya, T.; Wada, H.; Uemura, K.; Shimatsu, A.; Kimura, T.; Fujita, M.; Hasegawa, K. Circulation 2009, 120, S763. (50) Wongcharoen, W.; Phrommintikul, A. Int. J. Cardiol. 2009, 133, 145. (51) Hong, D. S.; Zeng, X. W.; Xu, W.; Ma, J.; Tong, Y. H.; Chen, Y. Pharmacol. Res. 2010, 61, 142. (52) Urry, D. W.; Haynes, B.; Harris, R. D. Biochem. Biophys. Res. Commun. 1986, 141, 749. (53) Yamaoka, T.; Tamura, T.; Seto, Y.; Tada, T.; Kunugi, S.; Tirrell, D. A. Biomacromolecules 2003, 4, 1680. (54) Christensen, T.; Amiram, M.; Dagher, S.; Trabbic-Carlson, K.; Shamji, M. F.; Setton, L. A.; Chilkoti, A. Protein Sci. 2009, 18, 1377. (55) Reiersen, H.; Clarke, A. R.; Rees, A. R. J. Mol. Biol. 1998, 283, 255. (56) Girotti, A.; Reguera, J.; Arias, F. J.; Alonso, M.; Testera, A. M.; ́ Rodriguez-Cabello, J. C. Macromolecules 2004, 37, 3396. (57) Yucel, T.; Micklitsch, C. M.; Schneider, J. P.; Pochan, D. J. Macromolecules 2008, 41, 5763. (58) Pochan, D. J.; Schneider, J. P.; Kretsinger, J.; Ozbas, B.; Rajagopal, K.; Haines, L. J. Am. Chem. Soc. 2003, 125, 11802.

Furthermore, it can be possible to exploit the Tt for localization of the nanoparticle delivery vehicle within a particular region via hyperthermia.13−16 Experiments are underway to explore the applications of these protein polymers for such biomedical applications.

■

ASSOCIATED CONTENT

S Supporting Information *

DNA gels, secondary structural analysis via CDSSTR, TEM analysis, DTT experiments, and fluorescence binding plots. This material is available free of charge via the Internet at http://pubs.acs.org.

■ ■

AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected].

ACKNOWLEDGMENTS This work was supported by AFOSR FA-9550-07-1-0060 and FA9550-08-1-0266 (J.K.M.), partially by the NSF MRSEC Program under award number DMR-0820341, Society of Plastic Engineers (J.S.H.), and GK-12 Fellows grant DGE-0741714 (J.S.H.).

■

REFERENCES

(1) Channon, K. J.; Devlin, G. L.; MacPhee, C. E. J. Am. Chem. Soc. 2009, 131, 12520. (2) John, G.; Vemula, P. K. Soft Matter 2006, 2, 909. (3) Kopecek, J. J. Polym. Sci., Part A: Polym. Chem. 2009, 47, 5929. (4) Shen, X. C.; Mo, X. Q.; Moore, R.; Frazier, S. J.; Iwamoto, T.; Tomich, J. M.; Sun, X. Z. S. J. Nanosci. Nanotechnol. 2006, 6, 837. (5) Connor, R. E.; Tirrell, D. A. Polym. Rev. 2007, 47, 9. (6) Voloshchuk, N.; Montclare, J. K. Mol. BioSyst. 2010, 6, 65. (7) Haghpanah, J. S.; Yuvienco, C.; Civay, D. E.; Barra, H.; Baker, P. J.; Khapli, S.; Voloshchuk, N.; Gunasekar, S. K.; Muthukumar, M.; Montclare, J. K. ChemBioChem 2009, 10, 2733. (8) Haghpanah, J. S.; Yuvienco, C.; Roth, E. W.; Liang, A.; Tu, R. S.; Montclare, J. K. Mol. Biosyst. 2010, 6, 1662. (9) Krishnaji, S. T.; Huang, W.; Rabotyagova, O.; Kharlampieva, E.; Choi, I.; Tsukruk, V. V.; Naik, R.; Cebe, P.; Kaplan, D. L. Langmuir 2011, 27, 1000. (10) Rabotyagova, O. S.; Cebe, P.; Kaplan, D. L. Macromol. Biosci. 2010, 10, 49. (11) Xu, C.; Breedveld, V.; Kopeček, J. Biomacromolecules 2005, 6, 1739. (12) Xu, C.; Kopeček, J. Pharm. Res. 2008, 25, 674. (13) Furgeson, D. Y.; Dreher, M. R.; Chilkoti, A. J. Controlled Release 2006, 110, 362. (14) Mackay, J. A.; Chilkoti, A. Int. J. Hyperthermia 2008, 24, 483. (15) Dreher, M. R.; Simnick, A. J.; Fischer, K.; Smith, R. J.; Patel, A.; Schmidt, M.; Chilkoti, A. J. Am. Chem. Soc. 2008, 130, 687. (16) Dreher, M. R.; Liu, W. G.; Michelich, C. R.; Dewhirst, M. W.; Chilkoti, A. Cancer Res. 2007, 67, 4418. (17) Nagarsekar, A.; Crissman, J.; Crissman, M.; Ferrari, F.; Cappello, J.; Ghandehari, H. J. Biomed. Mater. Res. 2002, 62, 195. (18) Nagarsekar, A.; Crissman, J.; Crissman, M.; Ferrari, F.; Cappello, J.; Ghandehari, H. Biomacromolecules 2003, 4, 602. (19) Dandu, R.; Von Cresce, A.; Briber, R.; Dowell, P.; Cappello, J.; Ghandehari, H. Polymer 2009, 50, 366. (20) Shen, W.; Kornfield, J. A.; Tirrell, D. A. Soft Matter 2007, 3, 99. (21) Shen, W.; Kornfield, J. A.; Tirrell, D. A. Macromolecules 2007, 40, 689. (22) Meyer, D. E.; Chilkoti, A. Biomacromolecules 2004, 5, 846. (23) Urry, D. W.; Mitchell, L. W.; Ohnishi, T. Biochim. Biophys. Acta, Protein Struct. 1975, 393, 296. (24) Baker, P. J.; Haghpanah, J. S.; Montclare, J. K. Elastin Based Protein Polymers. In Green Polymer Chemistry: Biocatalysis and 4245

dx.doi.org/10.1021/bm201083d | Biomacromolecules 2011, 12, 4240−4246

Biomacromolecules

Article

(59) Kretsinger, J. K.; Haines, L. A.; Ozbas, B.; Pochan, D. J.; Schneider, J. P. Biomaterials 2005, 26, 5177. (60) Morihara, Y.; Ogata, S.-I.; Kamitakahara, M.; Ohtsuki, C.; Tanihara, M. J. Polym. Sci., Part A: Polym. Chem. 2005, 43, 6048. (61) Collier, J. H.; Hu, B. H.; Ruberti, J. W.; Zhang, J.; Shum, P.; Thompson, D. H.; Messersmith, P. B. J. Am. Chem. Soc. 2001, 123, 9463. (62) Betre, H.; Liu, W.; Zalutsky, M. R.; Chilkoti, A.; Kraus, V. B.; Setton, L. A. J. Controlled Release 2006, 115, 175.

4246

dx.doi.org/10.1021/bm201083d | Biomacromolecules 2011, 12, 4240−4246