Influence of Fluorination on Protein-Engineered Coiled-Coil Fibers

Mar 20, 2015 - The fluorinated proteins C+TFL and Q+TFL are highly α-helical as confirmed via circular dichroism (CD) and more resistant to thermal d...
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Influence of Fluorination on Protein-Engineered Coiled-Coil Fibers Haresh T. More,† Kevin S. Zhang,† Nikita Srivastava,† Joseph A. Frezzo,† and Jin K. Montclare*,†,∫ ,§ †

Department of Chemical and Biomolecular Engineering, New York University Polytechnic School of Engineering, Brooklyn, New York 11201, United States ∫ Department of Biochemistry, SUNY Downstate Medical Center, Brooklyn, New York 11203, United States § Department of Chemistry, New York University, New York, New York 10003, United States S Supporting Information *

ABSTRACT: We describe the design and characterization of fluorinated coiled-coil proteins able to assemble into robust nanoand microfibers. Fluorination is achieved biosynthetically by residue-specific incorporation of 5,5,5-trifluoroleucine (TFL). The fluorinated proteins C+TFL and Q+TFL are highly α-helical as confirmed via circular dichroism (CD) and more resistant to thermal denaturation compared to their nonfluorinated counterparts, C and Q. The fluorinated proteins demonstrate enhanced fiber assembly at pH 8.0 with higher order structure in contrast to nonfluorinated proteins, which are unable to form fibers under the same conditions. Ionic strength dependent fiber assembly is observed for fluorinated as well as wild-type proteins in which the fluorinated proteins exhibited more stable, thicker fibers. The fluorinated and nonfluorinated proteins reveal metal ion-dependent small molecule recognition and supramolecular assemblies. In the presence of Zn (II), enhanced thermal stability and fiber assembly is observed for the fluorinated proteins and their nonfluorinated counterparts. Whereas Ni (II) promotes aggregation with no fiber assembly, the stabilization of α-helix by Zn (II) results in enhanced binding to curcumin by the fluorinated proteins. Surprisingly, the nonfluorinated proteins exhibit multiplefold increase in curcumin binding in the presence of Zn (II). In the context of the growing number of protein-based fiber assemblies, these fluorinated coiled-coil proteins introduce a new paradigm in the development of highly stable, robust selfassembling fibers under more physiologically relevant pH conditions that promotes the binding and release of small molecules in response to external cues.

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INTRODUCTION In nature, biological macromolecules are programmed to selfassemble in ordered arrays, leading to a complex machinery capable of sophisticated functions.1 The structural investigation of these implicit biological functions and their supramolecular assemblies serve as a starting point to engineer the novel nanoscale structures with well-defined architecture. Naturally, higher-order self-assembly has been observed in many different proteins including collagen,2−4 keratin,5 vimetin,6 fibrinogen,7,8 actin,9 optically active reflectin,10 cystallins11 and amyloid fibrils.12 The α-helical coiled-coil protein-based architecture has been explored by several researchers to develop different fibril assemblies with a size spanning from the nanometer to micrometer scale.13,14 We15,16 and others17 have developed pentameric coiled-coil assemblies forming fibers of 10 nm to 16 μm in diameter. Linear coiled-coil assemblies have been developed by Kojima et al. based on α3-peptide system that self-assembles into a fiber with diameter of 5−10 nm.18 Headto-tail sticky end self-assembling heterodimers (SAF) have been constructed by Woolfson and co-workers13,19−21 to form fibers with thickness of 10−100 nm. Blunt end coiled-coil peptides © 2015 American Chemical Society

have been developed by the Hartgerink group to assemble into fibers with 4−20 nm diameter.22 As an alternative strategy, a 3helix bundle protein (A−C) in which the C domain is removed and attached as an extension to the B domain resulted in fibers with diameters of 40−70 nm consisting of several protofibrils.22,23 In these cases, hydrophobic residues such as Leu, Ile, and Val are engineered at the interfaces of coiled-coil proteins. Integrating fluorinated amino acids in peptide and proteins have been demonstrated to improve stability.24−29 In particular, trifluoroleucine,27 trifluorovaline,28,30 trifluoroisoleucine30 and hexafluoroleucine31 have been incorporated into coiled-coils, leading to stability against heat and chemical denaturation as well as higher propensity for oligomerization. While extensive studies have been performed on helical proteins (vide supra), the impact of fluorination on fiber assembly has not been explored. Here we have engineered and characterized fluorinated coiled-coil protein fibers capable of: (i) binding and Received: December 31, 2014 Revised: March 17, 2015 Published: March 20, 2015 1210

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centrifuged, and the supernatant was further purified by fast protein liquid chromatography (FPLC) using HiTrap IMAC FF (5 mL, GE life sciences), which was charged with 100 mM CoCl2 and subsequently equilibrated with lysis buffer. The protein was eluted by increasing buffer B (6 M urea, 50 mM Na2HPO4, 1 M imidazole, pH 8.0) concentration from 2% and 100% v/v. Fractions of 5 mL were collected and ran on SDS-PAGE. These purified proteins were stepwise dialyzed using 3 M, 1.5 and 0.75 M of urea in 50 mM phosphate buffer pH 8.0 and three buckets of 50 mM phosphate buffer pH 8.0. Trifluoroleucine Incorporation Analysis. The incorporation of TFL was assessed by matrix-assisted laser desorption ionization, timeof-flight (MALDI-TOF) mass spectrometry analysis on UltrafleXtreme Mass Spectrometer (Bruker). For trypsin digest, 2 μL of sequencing grade modified trypsin was added to 20 μL of 0.5 mg/mL purified protein in 50 mM phosphate buffer pH 8.0 and incubated for 6 h at 37 °C. The reaction was quenched by addition of 2 μL of 10% trifluoroacetic acid (TFA). The sample was subjected to zip-tip using C18 packed tips (EMD Millipore). The tips were equilibrated with 75% acetonitrile (ACN) and eluted with 0.1% TFA in 75% ACN and mixed at a 1:1 ratio with saturated CCA matrix dissolved in 50% ACN with 1% TFA. Samples were spotted on Bruker MALDI target plate and air-dried. Theoretical trypsin digest of proteins were performed using Expasy PeptideMass online tool (http://web.expasy.org/ peptide_mass/). The mass spectra were analyzed for relative incorporation in C+TFL and Q+TFL protein. The incorporation of TFL was further confirmed by amino acid analysis (AAA) facilities at W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University. Protein Fiber Assembly. The fiber assembly of C+TFL, Q+TFL and nonfluorinated counterparts was evaluated using 10 μM of protein in 50 mM phosphate buffer pH 8.0. Also the effect of sodium chloride on fiber formation was analyzed at 100 mM and 500 mM salt concentration. Metal ion-induced changes in the fiber assembly were observed with all proteins in the presence of 100 μM of Ni (II) and Zn (II). The protein solutions were incubated at 4 °C without agitation and monitored by transmission electron microscopy (TEM) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). Circular Dichroism Spectroscopy. The secondary structure was assessed on 10 μM of proteins concentration, and the circular dichroism (CD) spectra was recorded using Jasco J-815 spectrometer attached with PTC-423S single position Peltier temperature control system. Wavelength scans were performed at 4 °C over a range of 250−200 nm with 1 nm step size using 1 mm quartz cuvette. Temperature scans were performed at 222 nm from 20 to 85 °C, at a heating rate of 1 °C/min. Mean residue ellipticity, fraction folded and melting temperature was calculated according to equations explained elsewhere.34 The α-helix, β-sheet, and random coil content from the secondary structure of proteins was analyzed using the K2D method using DichroWeb software.39,40 The effect of hydrophobic fluorinated solvent on the protein structure was evaluated with increasing concentration of TFE from 5% v/v to 40% v/v in 50 mM phosphate buffer pH 8.0. Proteins were incubated for 24 h at 4 °C prior to CD studies. For impact of soluble metal ions study, 10 μM of fluorinated and nonfluorinated proteins were mixed with 100 μM of Zn (II)/Ni (II) and incubated for 24 h at 4 °C. Transmission Electron Microscopy. The electron micrographs of protein fiber were acquired using Zeiss EM-902 TEM. Approximately 3 μL of 10 μM protein sample was applied on a 400 mesh copper grid and after 1 min, the excessive solution was blotted out by filter paper. The grid was washed twice with dH2O, negatively stained with 3 μL of 1% w/v uranyl acetate for 1 min, and excess solution was blotted out with filter paper and air-dried at room temperature. The dimensions of the fibers were analyzed by ImageJ software.41 Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy. The ATR-FTIR studies on protein samples were performed using PerkinElmer System 2000 FT-IR with DuraSamplIR II T diamond ATR accessory and equipped with a MCT A detector.

encapsulating a guest small molecule; and (ii) responding to external soluble metal ions. These intelligent fluorinated fibers demonstrate metal-dependent binding in which Zn (II) stabilizes the coiled-coil structure and fiber formation to improve small molecule encapsulation and Ni (II) promotes aggregation, effectively eliminating small molecule recognition. In contrast to their nonfluorinated counterparts, the fluorinated proteins assemble into fibers at pH 8.0, indicating that incorporation of the fluorinated amino acid improves structure and self-assembly. Moreover, the fluorinated coiled-coils form more stable, thicker fibers; the fluorination is crucial not only to the formation of robust fibers but also stability overall.

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EXPERIMENTAL SECTION

Materials. Isopropyl-β-D-1-thiogalactopyranoside (IPTG), ampicillin, kanamycin, thymine hydrochloride (vitamin B), tryptic soy agar and urea were purchased from Fisher. All 20 amino acids were obtained from Sigma-Aldrich and racemic 5,5,5-trifluoroleucine (TFL) from Oakwood chemical. Potassium dihydrogen phosphate, calcium chloride, magnesium sulfate, disodium hydrogen phosphate, ammonium chloride, glucose monohydrate, nickel(II) chloride hexahydrate, zinc chloride and α-cyano-4-hydrocinnamic acid (CCA) was purchased from Sigma-Aldrich. Methanol, acetonitrile, trifluoroacetic acid, 2,2,2-trifluoroethanol (TFE) and curcumin (CCM) from Acros Organics, imidazole from Alfa Aesar, trypsin from Promega BCA kit Pierce from VWR and 400 mesh carbon supported copper TEM grids from Electron Microscopy Sciences. Protein Models. The protein models were generated using the available crystal structure of cartilage oligomeric matrix protein coiledcoil (COMPcc) (1VDF).32 The protein structure was visualized, and residues were mutated using Chimera molecular modeling software.33 The parent COMPccs was mutated at C68S and C71S residues to produce COMPcc,34 and this was used as a template to construct C and Q.16 The Leu residues were mutated into TFL using SwissSide chain viewer plugin enabled in Chimera. The rotamer libraries generated in SwissSide chain were chosen to select the rotamer with highest probability for each TFL residue substituted at given position.35,36 The electrostatic surface map was generated after substituting all Leu residues with TFL and considering pentameric self-assembly of five monomer units to produce C+TFL and Q+TFL. Protein Expression. Leu auxotrophic Escherichia coli strain LAM1000 cells37,38 were used for incorporation of TFL in C and Q protein via residue-specific incorporation. The pQE30/C and pQE30/ Q16 plasmids were transformed by electroporation, and colonies were grown on tryptic soy agar plate with ampicillin (200 μg/mL) and kanamycin (35 μg/mL). Starter cultures were prepared in 4 mL of 1xM9 minimal media supplemented with 20 amino acids, ampicillin (200 μg/mL), and kanamycin (35 μg/mL) and incubated at 37 °C at 250 rpm. For each protein, a 200 mL expression was performed in a shake flask with the above-mentioned supplements. The cells were grown at optical density at 600 nm (OD) of 1.0, pelleted, and extensively washed with ice cold 0.9% w/v NaCl three times for complete removal of amino acids. The cell pellets were resuspended in new M9 minimal media containing 19 amino acids except leucine and all other ingredients previously mentioned. The cells were grown for another 20 min at 37 °C at 250 rpm to completely deplete leucine, and protein expression was induced by addition of IPTG (200 μg/mL) in the presence of TFL (555 μg/mL) and incubated at the same conditions for 3 h. Cells were harvested after overexpression by centrifugation and stored at −80 °C until purification. The expression of C+TFL and Q+TFL was confirmed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Purification. The fluorinated proteins as well as their nonfluorinated counterparts were purified under denaturing conditions. The cell pellets were thawed at 4 °C for 30 min and resuspended in lysis buffer (6 M urea, 50 mM Na2HPO4, 20 mM imidazole, pH 8.0) and lysed using ultrasonic probe sonicator (Q500 sonicator,amplitude 40%, pulse 5 s on and 5 s off, time −2 min). Whole cell lysates were 1211

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Figure 1. Protein design and electrostatic surface rendering of fluorinated proteins. (a) Protein sequence for C and Q with Leu residues highlighted that was replaced with TFL to produce C+TFL and Q+TFL respectively. (b) Surface charge of C+TFL (left) and Q+TFL (right); blue represents positive and red represents negative surface. (c) SDS-PAGE confirmation of proteins overexpression in the absence and presence of TFL.

Figure 2. (a) CD wavelength scan of C+TFL (solid line), Q+TFL (gray line), C (black dashed line), and Q (gray dashed line); (b) conformational transition of proteins observed at various concentration of TFE measured as MRE at 222 nm, C+TFL (diamonds solid line), Q+TFL (circle gray line), C (diamond black dashed line), Q (circle gray dashed line). Protein concentration is at 10 μM in 50 mM phosphate buffer, pH 8.0, 4 °C.

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Approximately 10 μL of protein solution was added onto the diamond ATR crystal surface. The spectrum was measured over a range of 4000−400 cm−1 with 0.5 cm−1 resolution with total 128 scans per measurement. The data was processed using PeakFit software,15 where the baseline correction was performed for amide I region between 1700 and 1600 cm−1 and peaks deconvoluted by Gaussian function. Fluorescence Studies. The binding of CCM to fluorinated and nonfluorinated proteins was carried out in 96-well black plate (Costar, Corning). For binding studies, total 10 μM of protein (50 mM PB, pH 8.0) was mixed with 100 μM of metals ions (Ni (II) and Zn (II)) and equilibrated for 24 h at 4 °C. CCM was added to the proteins to a final concentration of 50 μM [50 mM PB (pH 8.0) and methanol (0.5%)] in the absence and presence of metals and incubated for 2 h at room temperature. Fluorescence was measured using a Synergy HT instrument. The CCM sample was excited at 420 nm and emission wavelength spectrum was measured over a range of 450−600 nm.34 The fluorescence peak maxima at 525 nm were used for evaluation of binding efficiency. The buffer in the absence and presence of metals upon binding to CCM did not produce any fluorescence around 525 nm.

RESULTS AND DISCUSSION Design Principles. Two fiber forming coiled-coil proteins C and Q were designed (Figure 1). While Q was previously developed and confirmed to assemble into fibers,16 C was engineered for this work and not previously explored. Based on COMPccs, the electrostatic surface map revealed a negative charge on the C-terminal heptad starting at Val65. Thus, the last heptad was removed to generate C, leading to uniform charge separation (Supporting Information Figure S1). As the distribution of surface charge is necessary for lateral assembly of protofibrils,21,22 The homopentameric assembly of C+TFL and Q+TFL was explored via Chimera.33 The fluorinated protein showed significantly different surface charges (Figure 1); highly positive surface charge was observed at the N-terminus of Q+TFL due to solvent-exposed Arg and Lys residues along with a negative patch toward the C-terminus due to Glu, Asp, Gln, and Asn residues (Figure 1b). In the case of C+TFL, charge is more scattered with a positive charged 1212

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Figure 3. TEM images of (a) C+TFL and (b) Q+TFL in the presence of 0 mM NaCl, 100 mM NaCl, and 500 mM NaCl in 50 mM phosphate buffer pH 8.0. The scale bars in the image are 200 nm and insets are 1 μm.

structure was observed at 10 and 20% v/v TFE (Figure 2b). For the nonfluorinated proteins, while C showed minimal changes in helicity, a pronounced effect was observed for the Q protein (Figure 2b). This could be attributed to the more helical structure of C, than to Q, which was predominantly unstructured in the absence of TFE (Figure S4, Table S3). Surprisingly at 40% v/v TFE, the C+TFL and Q+TFL demonstrated an increase in helicity with an overall 86% and 85% helical content, respectively (Figure 2b, Table S3. Moreover, C and Q showed a further increase in structure with a 74% and 79% helical content, respectively (Figure 2b, Table S3). The induction of α-helical structure in nonfluorinated proteins was mainly due to the stabilization of intermolecular and intramolecular hydrogen bonds presumably due to their low dielectric constant; the bulky trifluoromethyl group of TFE was unable to interact with peptide backbone.42 At concentrations of up to 20% v/v TFE, the trifluoromethyl groups interacted with each other, solvating them, leading to a decrease in structure for the fluorinated proteins. At a higher concentration of 40% v/v TFE, the amide backbone desolvation dominated, sharply increasing the helicity of the fluorinated as well nonfluorinated proteins,28,42 consistent with previous studies reporting that 30−40% v/v TFE led to enhanced helical structure of the proteins.43 Visualization of Fiber Assembly and Structure in Solid State. To determine whether the fluorinated and nonfluorinated proteins assembled into fibers, TEM was employed. Both fluorinated proteins exhibited fiber assembly at pH 8.0, consistent with the proposed model for protofibrils and mature fiber assembly due to electrostatic interactions of fluorous corestabilized pentameric structures (Figure 3, Figure S5). The C +TFL and Q+TFL assembled into fibers with diameters from 39 nm to 2.0 μm (Figure 3a, S6, Table S3) and 42 nm to 1.5 μm in size (Figure 3b, S7, Table S4), respectively. Upon higher magnification, both revealed bundles of protofibrils with a diameter of 3.6 ± 0.3 nm, arranged in lateral as well as longitudinal assembly to form mature fibers (Figure 3). In contrast to the fluorinated proteins, the nonfluorinated proteins did not assemble into fibers at pH 8.0 (Figure S8). While the TFL residues were predominantly buried in the hydrophobic core, the model revealed that the C-terminal TFL residues of

surface near the C-terminus due to solvent-exposed lysine and arginine residues, whereas a small negative surface patch is observed near the center due to solvent-exposed Asp and Glu residues from second and third heptad repeats. Biosynthesis of Proteins. The expression of fluorinated and nonfluorinated proteins via Leu auxotrophic Escherichia coli strain LAM 100038 was confirmed by SDS-PAGE in the presence and absence of TFL and/or Leu (Figure 1c). In the presence of Leu or TFL, an overexpression band at 6.3 kDa was observed for C, Q, C+TFL, and Q+TFL (Figure 1c). Upon purification, the levels of TFL incorporation were assessed via MALDI-TOF mass spectrometry and amino acid analysis (Figure S2, S3, Table S1). Based on the MALDI-TOF analysis, the extent of TFL substitution was 91.9% for C+TFL and 94.7% for Q+TFL. These high levels of incorporation were confirmed by AAA, revealing incorporation levels of 95.1% for C+TFL and 90.7% for Q+TFL (Table S1). Secondary Structure and Stability Studies. To assess the impact of TFL on the secondary structure of the proteins, CD was performed on the fluorinated proteins and compared to their nonfluorinated counterparts in 50 mM PB pH 8.0 (Figure 2a). Both C+TFL and Q+TFL demonstrated highly helical conformations with a 78% and 58% helical content, respectively (Table S2). The nonfluorinated proteins revealed less structure with a 26% and 9% helical content for C and Q, respectively (Table S2). Thus, fluorination improved the helical structure for both C+TFL and Q+TFL by 3 and 6.4 fold, respectively (Figure 2a), consistent with previous studies.26−29 To determine the effect of fluorination on protein stability, the melting temperature (Tm) was obtained by monitoring the changes in ellipticity at 222 nm as a function of temperature. The C and Q proteins demonstrated similar stabilities with a Tm of 42 and 39 °C, respectively (Figure 2a, Table S2). Relative to the nonfluorinated proteins, C+TFL and Q+TFL exhibited a 14 and 13 °C increase in Tm, respectively (Figure 2a, Table S2). These results affirmed prior investigations by others indicating that fluorinated coiled-coils improved stability.26−29 The helix stabilizing interactions by the TFL was further evaluated in the presence of TFE. The effect of TFE on helicity was determined by assessing the signal change at 222 nm. In the case of the fluorinated proteins, a drastic decrease in 1213

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demonstrated that engineered coiled-coils of COMPccs variants were responsive to soluble divalent Ni (II) and Zn (II) metal ions due to the presence of an N-terminal hexahistidine-tag.15 Thus, we sought to investigate the impact of divalent metal ions on the structure and stability of the engineered fluorinated proteins and their nonfluorinated counterparts. Remarkably, C +TFL revealed large fibers with diameters ranging from 16 nm to 2.2 μm that further assembled into sheet-like structures in the presence of Zn (II) (Figure 4a, S16, Table S11). In the case of Q+TFL, fibers with thickness of 31−553 nm (Figure 4b, S17, Table S12) was observed with Zn (II), similar to conditions in the absence of metal ions (Figure 3b). Surprisingly, the nonfluorinated proteins demonstrated fiber assembly in the presence of Zn (II), where C presented fibers with diameter of 40−546 nm (Figure 4c, S18, Table S13) and Q demonstrated fibers with 69−569 nm thickness (Figure 4d, S19, Table S14). The fluorinated and nonfluorinated proteins assembled into aggregate structures in the presence of Ni (II). Overall, Zn (II) facilitated fiber assembly, while Ni (II) did not, consistent with prior studies.15 Similar behavior was observed by Pagel et al., where amyloid forming coiled-coil peptides was unfolded, leading to aggregates in the presence of Cu (II), which possessed a specificity for His, whereas, in the presence of Zn (II), amyloid fiber formation was promoted.45 The assemblies in the presence/absence of metal ions were assessed for structure and stability in solution. In the presence of 100 μM Zn (II), both C+TFL and Q+TFL showed characteristic double minima at 222 and 208 nm with a 71% and 59% helicity, respectively (Figure 5a, Table S15). Both proteins showed drastic increase in thermal stability (Tm > 80 °C), confirming that Zn (II) stabilized the structures (Figure 5a). By contrast, addition of 100 μM Ni (II) to C+TFL and Q +TFL led to a complete loss of helical structure and an observed minima at 228 nm, indicative of higher order aggregates as previously observed with wt COMPccs 15 (Figure 5b, Table S15). Both the nonfluorinated C and Q showed significant change in secondary structure with enhanced minima at 208 and 222 nm; temperature-dependent studies revealed a Tm of >80 °C for C and 65 °C for Q in the presence of 100 μM Zn (II) (Figure 5b, Table S15). In the presence of 100 μM Ni (II), a drastic decrease in the minima at 208 and red shift of the 222 nm minima to 228 nm occurred for C and Q proteins, indicative of large aggregates as observed with fluorinated proteins (Figure 5b, Table S15). The secondary structure of the proteins within the supramolecular assemblies in solid state was determined via ATR-FTIR. In the presence of Zn (II), protein fibers exhibited a major peak at 1652 cm−1 indicative of helical conformation,46 with an increase in helical content of 79% for C+TFL and 78% for Q+TFL (Figure S15, Table 1). The nonfluorinated proteins also revealed α-helical structure with a 71% and 39% helical content for C and Q upon addition of Zn (II), respectively (Figure S15, Table 1). ATR-FTIR spectra showed higher order aggregates for all proteins in the presence of Ni (II) demonstrating peaks between 1618 cm−1 and 1624 cm−1, with an overall loss of α-helical conformation (Figure S15, Table 1) and increase in β-sheet and random structures. These data were consistent with the solution studies and conformational analysis. The design of the proteins maintained the hydrophobic residues at a and d position in order to keep the pore intact for binding to small molecules. Although the secondary structure analysis showed loss in helicity for the nonfluorinated proteins

both C+TFL and Q+TFL were solvent exposed (Figure 1b). Fibrous sheet-like structures were observed for both fluorinated proteins presumably due to the C-terminal TFL residues, providing hydrophobic interactions among the assembled pentameric structures, leading to protofibril formation. The impact of surface charge was investigated to assess whether the electrostatic interactions drove protofibril assembly, resulting in the overall fiber structure. Surface charge screening was carried out using NaCl at 100 mM and 500 mM concentrations. While C+TFL revealed sheet-like structures with diameter of 114−695 nm (Figure 3a, S9, Table S5), Q +TFL showed longer and robust fiber assembly at 100 mM NaCl (Figure 3b, S10, Table S6). At 500 mM NaCl, C+TFL demonstrated shorter fiber assembly with diameter of 14−166 nm, where larger aggregates were observed for Q+TFL (Figure 3a, S11, Table S7). In contrast to no salt conditions where fibers were not observed for both nonfluorinated proteins, C illustrated fibers of 98−968 nm diameters at 100 mM NaCl (Figure S8, S12, Table S8) and 10−15 nm at 500 mM NaCl (Figure S8, S13, Table S9), while Q showed more uniform long fibers of 38−162 nm thickness at 100 mM NaCl (Figure S8, S14, Table S10) and large aggregates at 500 mM NaCl (Figure S8). Overall, both the fluorinated C+TFL and Q+TFL proteins exhibited more stable, thicker fibers when compared to their nonfluorinated counterparts. In order to determine whether the fibers themselves were αhelical in the solid state, ATR-FTIR was performed on the fluorinated and nonfluorinated proteins (Table 1). Both C Table 1. ATR-FTIR Summary of Secondary Structure Composition of Fiber Assembly of C+TFL and Q+TFL, C and Q Proteins in the Presence and Absence of Metal Ionsa % composition protein

α-helix

β-sheet

random coil

C C/Ni (II) C/Zn (II) C+TFL C+TFL/Ni (II) C+TFL/Zn (II) Q Q/Ni (II) Q/Zn (II) Q+TFL Q+TFL/Ni (II) Q+TFL/Zn (II)

62 30 71 77 31 79 37 19 39 71 32 78

38 45 29 23 39 21 33 25 29 29 37 22

25 30 30 41 32 31 -

Percent composition was determined from relative areas of peaks fit to spectra (Figure S15). a

+TFL and Q+TFL showed characteristic α-helical bands in the amide I and amide II regions, specifically in the 1650−1653 cm−1 region (Figure S15). A broad peak with multiple components was observed in the amide I region, indicative of coiled-coil assembly.44 Deconvoluted data of C+TFL and Q +TFL protein fibers revealed an α-helical content of 77% and 71%, respectively (Figure S8, Table 1). The C and Q nonfibrous proteins possessed less helical values of 62% and 37%, respectively; they were less structured than fluorinated counterparts (Figure S15, Table 1). Hence the fluorinated protein fibers were indeed comprised of helical assemblies. Controlling the Fiber Assemblies and Guest Binding via Soluble Divalent Metals. Our previous studies 1214

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Figure 4. TEM images of (a) C+TFL, (b) Q+TFL, (c) C, and (d) Q in the presence of 100 mM Ni (II) and 100 mM Zn (II) in 50 mM phosphate buffer pH 8.0. The scale bars in the image are 200 nm, and the insets are 1 μm.

Figure 5. CD wavelength scans of (a) C+TFL, Zn (II) [solid line] and Q+TFL, Zn (II) [gray line] and in the presence of Ni (II) [dashed black and gray line], respectively, and (b) C, Zn (II) [solid line] and Q, Zn (II) [gray line] and in the presence of Ni (II) [dashed black and gray line] respectively. (c) Curcumin binding to fluorinated and nonfluorinated proteins in presence and absence of Ni (II) and Zn (II).

C and Q at pH 8.0, the fluorinated proteins C+TFL and Q +TFL demonstrated enhanced helical structure. The binding ability of the fluorinated and nonfluorinated proteins to CCM was assessed in the presence or absence of divalent metal ions.15,34 In absence of metal ions, the parent nonfluorinated C revealed minimal binding for CCM, whereas Q did not bind CCM (Figure 5c). Both fluorinated proteins bound CCM with an observed fluorescence signal of 2011 ± 170 RFU for C+TFL and 1906 ± 122 RFU for Q+TFL, respectively. The fluorinated proteins demonstrated enhanced binding to CCM when

compared to the nonfluorinated counterparts, presumably due to higher helical content and maintenance of the hydrophobic pore indispensible for small molecule binding (Table 1, S15). In the presence of 100 μM Zn (II), a 1.5−2 fold increase in fluorescence signal for C+TFL and Q+TFL was observed, suggesting significantly enhanced binding to CCM (Figure 5c). Surprisingly, the nonfluorinated proteins revealed binding to CCM in the presence of Zn (II) with a fluorescence signal of 8527 ± 455 RFU for C and 23 386 ± 804 RFU for Q (Figure 5c). This ability to recognize CCM was due to stabilization of 1215

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Biomacromolecules the α-helical conformation as demonstrated by CD where C and Q exhibited 2.4 and 5 fold increase in helical content, respectively (Figure 5b, Table S15). In the presence of Ni (II), binding to CCM was abolished for the fluorinated and nonfluorinated proteins (Figure 5c). Thus, loss in CCM recognition was in large part due to the formation of aggregates, as demonstrated by the strong negative minima at 228 nm.15 Although fluorinated proteins demonstrated increased CCM fluorescence in the presence of Zn (II), multifold increase in fluorescence intensity was observed for nonfluorinated proteins. It could be due to the steric constraint created by the trifluoromethyl group of TFL, which is twice the size of the trifluoromethyl group of Leu residues.47 This increase in hydrodynamic volume of trifluoromethyl group inside C+TFL and Q+TFL hydrophobic pores restricted the free movement of CCM relative to the nonfluorinated proteins.48 In addition, the electronegativity of fluorine atom could presumably affect the extrinsic fluorescence properties of CCM leading to changes in fluorescence intensity.49,50 Interestingly, while the nonfluorinated proteins were unable to form fibers, it was possible to trigger fiber assembly and improve binding capacity to CCM.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

S Supporting Information *

Supporting Information includes the figure for surface charge of wt COMPccs protein and schematic of fiber assembly, SDSPAGE of purified protein, MALDI-TOF spectra, AAA table, CD wavelength scan of nonfluorinated proteins and TEM images of nonfluorinated proteins, ATR-FTIR spectra at different solution conditions, and summary of CD data. This material is available free of charge via the Internet at http:// pubs.acs.org. Corresponding Author

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

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The author thanks Chin Lin (NYU shared instrument facility) and Jorge Morales (TEM facility CCNY) for their technical assistance for the experiments. This work was supported by ARO (W911NF-10-1-0228) and (W911NF-11-1-0449) (J.K.M.), NSF GK-12 fellow grant DGE-0741714 (J.A.F.), and in part by the NSF MRSEC Program under Award Number DMR-1420073 as well as the NYU CTSA Grant ULITR000038 from the National Center for Advancing Translational Sciences (NCATS), NIH. We dedicate this work to Professor Iwao Ojima for his 70th Birthday.

CONCLUSION

We have outlined a strategy for engineering fluorinated pentameric coiled-coil protein fiber assemblies. These genetically engineered fluorinated proteins assemble into robust nano- and microfibers under micromolar concentrations and are capable of binding the small molecule, CCM. They are responsive to external cues and can be programmed to both bind and release small molecule, alter fiber diameter and morphology in the presence of divalent metals. The fiber assembly is dependent on fluorination as when expressed under conventional 20 amino acids, the nonfluorinated counterparts are unable to form fibers at pH 8.0 due to inability to form highly helical conformations. This can be attributed to the improved stability and surface area for optimal assembly. Previous studies have been performed on rationally designed coiled-coil protein based self-assembling first, second and third generation fibers with alternate surface charge,21 different amino acid composition,19 additional heptad repeats.20 We demonstrate that fluorination of the hydrophobic core of engineered coiled-coil proteins can lead to robust fibers assembly. The fluorination not only enhances the overall stability and assembly of fiber, but also the binding of CCM due to its highly helical structure, whereas the nonfluorinated proteins fail to bind under the same conditions. The fluorinated proteins also show a unique dependence on metal ion15 based recognition of CCM and supramolecular assembly in fibers. The nanoscale assembly of fluorinated pentameric subunits into larger fibers at neutral pH and low concentration hold tremendous potential in biomaterials design with application in tissue engineering as well as drug delivery, as they can be visualized via nondestructive techniques such as 19F-NMR51 and MRI.52 Our studies demonstrate that protein-based fibrous assemblies can be designed through a bottom up approach in conjunction with fluorinated amino acids, which can further fine-tune the assemblies and provide opportunity to developed novel biomaterials with unique physicochemical properties.

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REFERENCES

(1) Zimenkov, Y.; Conticello, V. P.; Guo, L.; Thiyagarajan, P. Tetrahedron 2004, 60, 7237−7246. (2) Kotch, F. W.; Raines, R. T. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 3028−3033. (3) Yu, S. M.; Li, Y.; Kim, D. Soft Matter 2011, 7, 7927−7938. (4) Pins, G. D.; Christiansen, D. L.; Patel, R.; Silver, F. H. Biophys. J. 1997, 73, 2164−2172. (5) Rouse, J. G.; Van Dyke, M. E. Materials 2010, 3, 999−1014. (6) Parry, D. A.; Strelkov, S. V.; Burkhard, P.; Aebi, U.; Herrmann, H. Exp. Cell Res. 2007, 313, 2204−2216. (7) Koo, J.; Rafailovich, M. H.; Medved, L.; Tsurupa, G.; Kudryk, B. J.; Liu, Y.; Galanakis, D. K. J. Thromb. Haemostasis 2010, 8, 2727− 2735. (8) Wei, G.; Reichert, J.; Bossert, J.; Jandt, K. D. Biomacromolecules 2008, 9, 3258−3267. (9) Tojkander, S.; Gateva, G.; Lappalainen, P. J. Cell Sci. 2012, 125, 1855−1864. (10) Kramer, R. M.; Crookes-Goodson, W. J.; Naik, R. R. Nat. Mater. 2007, 6, 533−538. (11) Healy, J.; Wong, K.; Sawyer, E. B.; Roux, C.; Domigan, L.; Gras, S. L.; Sunde, M.; Larsen, N. G.; Gerrard, J.; Vasudevamurthy, M. Biopolymers 2012, 97, 595−606. (12) Serpell, L. C. Biochim. Biophys. Acta 2000, 1502, 16−30. (13) Pandya, M. J.; Spooner, G. M.; Sunde, M.; Thorpe, J. R.; Rodger, A.; Woolfson, D. N. Biochemistry 2000, 39, 8728−8734. (14) Ryadnov, M. G.; Woolfson, D. N. Nat. Mater. 2003, 2, 329−332. (15) Gunasekar, S. K.; Anjia, L.; Matsui, H.; Montclare, J. K. Adv. Funct. Mater. 2012, 22, 2154−2159. (16) Hume, J.; Sun, J.; Jacquet, R.; Renfrew, P. D.; Martin, J. A.; Bonneau, R.; Gilchrist, M. L.; Montclare, J. K. Biomacromolecules 2014, 15, 3503−3510. 1216

DOI: 10.1021/bm5019062 Biomacromolecules 2015, 16, 1210−1217

Article

Biomacromolecules

(49) Srinivas, V.; Santhoshkumar, P.; Krishna Sharma, K. J. Protein Chem. 2002, 21, 87−95. (50) Hawe, A.; Sutter, M.; Jiskoot, W. Pharm. Res. 2008, 25, 1487− 1499. (51) Danielson, M. A.; Falke, J. J. Annu. Rev. Biophys. Biomol. Struct. 1996, 25, 163−195. (52) Mehta, V. D.; Kulkarni, P. V.; Mason, R. P.; Constantinescu, A.; Antich, P. P. Bioconjugate Chem. 1994, 5, 257−261.

(17) Potekhin, S. A.; Melnik, T. N.; Popov, V.; Lanina, N. F.; Vazina, A. A.; Rigler, P.; Verdini, A. S.; Corradin, G.; Kajava, A. V. Chem. Biol. 2001, 8, 1025−1032. (18) Shuichi Kojima, Y. K.; Yoshida, T.; Yazaki, K.; Miura, K.-i. Proc. Jpn. Acad., Ser. B 1997, 73, 7−11. (19) Papapostolou, D.; Bromley, E. H. C.; Bano, C.; Woolfson, D. N. J. Am. Chem. Soc. 2008, 130, 5124−5130. (20) Smith, A. M.; Banwell, E. F.; Edwards, W. R.; Pandya, M. J.; Woolfson, D. N. Adv. Funct. Mater. 2006, 16, 1022−1030. (21) Papapostolou, D.; Smith, A. M.; Atkins, E. D.; Oliver, S. J.; Ryadnov, M. G.; Serpell, L. C.; Woolfson, D. N. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 10853−10858. (22) Dong, H.; Paramonov, S. E.; Hartgerink, J. D. J. Am. Chem. Soc. 2008, 130, 13691−13695. (23) Ogihara, N. L.; Ghirlanda, G.; Bryson, J. W.; Gingery, M.; DeGrado, W. F.; Eisenberg, D. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 1404−1409. (24) Baker, P. J.; Montclare, J. K. ChemBioChem 2011, 12, 1845− 1848. (25) Yuvienco, C.; More, H. T.; Haghpanah, J. S.; Tu, R. S.; Montclare, J. K. Biomacromolecules 2012, 13, 2273−2278. (26) Montclare, J. K.; Son, S.; Clark, G. A.; Kumar, K.; Tirrell, D. A. ChemBioChem 2009, 10, 84−86. (27) Tang, Y.; Ghirlanda, G.; Vaidehi, N.; Kua, J.; Mainz, D. T.; Goddard, I. W.; DeGrado, W. F.; Tirrell, D. A. Biochemistry 2001, 40, 2790−2796. (28) Bilgicer, B.; Fichera, A.; Kumar, K. J. Am. Chem. Soc. 2001, 123, 4393−4399. (29) Buer, B. C.; de la Salud-Bea, R.; Al Hashimi, H. M.; Marsh, E. N. G. Biochemistry 2009, 48, 10810−10817. (30) Son, S.; Tanrikulu, I. C.; Tirrell, D. A. ChemBioChem 2006, 7, 1251−1257. (31) Tang, Y.; Tirrell, D. A. J. Am. Chem. Soc. 2001, 123, 11089− 11090. (32) Malashkevich, V. N.; Kammerer, R. A.; Efimov, V. P.; Schulthess, T.; Engel, J. Science 1996, 274, 761−765. (33) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. J. Comput. Chem. 2004, 25, 1605−1612. (34) Gunasekar, S. K.; Asnani, M.; Limbad, C.; Haghpanah, J. S.; Hom, W.; Barra, H.; Nanda, S.; Lu, M.; Montclare, J. K. Biochemistry 2009, 48, 8559−8567. (35) Gfeller, D.; Michielin, O.; Zoete, V. Nucleic Acids Res. 2013, 41, D327−332. (36) Gfeller, D.; Michielin, O.; Zoete, V. J. Comput. Chem. 2012, 33, 1525−1535. (37) Voloshchuk, N.; Lee, M. X.; Zhu, W. W.; Tanrikulu, I. C.; Montclare, J. K. Bioorg. Med. Chem. Lett. 2007, 17, 5907−5911. (38) Panchenko, T.; Zhu, W. W.; Montclare, J. K. Biotechnol. Bioeng. 2006, 94, 921−930. (39) Whitmore, L.; Wallace, B. A. Biopolymers 2008, 89, 392−400. (40) Whitmore, L.; Wallace, B. A. Nucleic Acids Res. 2004, 32, W668−673. (41) Abramoff, M. D.; Magalhaes, P. J.; Ram, S. J. Biophotonics Int. 2004, 11, 36−42. (42) Schonbrunner, N.; Wey, J.; Engels, J.; Georg, H.; Kiefhaber, T. J. Mol. Biol. 1996, 260, 432−445. (43) Hong, D.-P.; Hoshino, M.; Kuboi, R.; Goto, Y. J. Am. Chem. Soc. 1999, 121, 8427−8433. (44) Heimburg, T.; Schünemann, J.; Weber, K.; Geisler, N. Biochemistry 1999, 38, 12727−12734. (45) Pagel, K.; Seri, T.; von Berlepsch, H.; Griebel, J.; Kirmse, R.; Böttcher, C.; Koksch, B. ChemBioChem 2008, 9, 531−536. (46) Pelton, J. T.; McLean, L. R. Anal. Biochem. 2000, 277, 167−176. (47) Leroux, F. ChemBioChem 2004, 5, 644−649. (48) Kwon, O.-H.; Yoo, T. H.; Othon, C. M.; Van Deventer, J. A.; Tirrell, D. A.; Zewail, A. H. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 17101−17106. 1217

DOI: 10.1021/bm5019062 Biomacromolecules 2015, 16, 1210−1217