Charge Reversal of the Rodlike Colloidal fd Virus through Surface

Care was taken to minimize intervirus cross linking, which often occurs ... rodlike viruses such as the tobacco mosaic virus (TMV), M13, and fd have b...
17 downloads 0 Views 2MB Size
pubs.acs.org/Langmuir © 2010 American Chemical Society

)

Charge Reversal of the Rodlike Colloidal fd Virus through Surface Chemical Modification

)

Zhenkun Zhang,†, Johan Buitenhuis,*,† Abhishek Cukkemane‡,^ Melanie Brocker,§ Michael Bott,§ and Jan K. G. Dhont† † IFF - Soft Condensed Matter, ‡ISB-1, and §IBT-1, Research Center J€ ulich, Germany. Current address: Department of Chemical Engineering, K.U. Leuven, Belgium. ^ Current address: NMR Spectroscopy Research Group, University Utrecht, The Netherlands

Received February 19, 2010. Revised Manuscript Received April 1, 2010 There is increasing interest in the use of viruses as model systems for fundamental research and as templates for nanomaterials. In this work, the rodlike fd virus was subjected to chemical modifications targeting different solventexposed functional groups in order to tune its surface properties, especially reversing the surface charge from negative to positive. The carboxyl groups of fd were coupled with different kinds of organic amines by carbodiimide chemistry, resulting in modified viruses that are positively charged over a wide range of pH. Care was taken to minimize intervirus cross linking, which often occurs because of such modifications. The surface amino groups were also grafted with poly(ethylene glycol) (PEG) end-functionalized with an active succinimidyl ester in order to introduce a steric stabilization effect. By combining charge reversal with PEG grafting, a reversible attraction between positively and negatively charged PEG-grafted fd viruses could be realized, which was tuned by the ionic strength of the solution. In addition, a charge-reversed fd virus forms only a pure nematic phase in contrast to the cholesteric phase of the wild type. These modified viruses might be used as model systems in soft condensed matter physics, for example, in the study of polyelectrolyte complexes or lyotropic liquid-crystalline phase behavior.

1. Introduction Because of their nearly perfect monodispersity in terms of size and shape, rodlike viruses such as the tobacco mosaic virus (TMV), M13, and fd have been used as model systems in soft condensed matter physics.1-4 Chemical modifications such as the introduction of fluorescent groups or the grafting of polymers onto the surface of the viruses have further increased the potential of these viruses in such applications.5-7 For example, poly(ethylene glycol) was grafted to the surface of the fd virus in order to control its effective diameter in the study of liquidcrystalline phase behavior,5 and dye-labeled viruses have often been used as tracers in a wide range of fundamental studies.7,8 Here we present work on the chemical modifications of fd viruses, demonstrating the possibility to reverse their charge over a wide range of pH, and show a few examples of possible applications of the modified viruses as model systems. Alternatively, we note that these or similar modifications might be used as precoatings to *Corresponding author. E-mail: [email protected] (1) Fraden, S. In Observation, Prediction, and Simulation of Phase Transitions in Complex Fluids; Baus, M., Rull, L., Ryckaert, J., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1995; Vol. 460, p 113. (2) Dogic, Z.; Fraden, S. In Soft Matter-Complex Colloidal Suspensions; Gompper, G., Schick, M., Eds.; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2006; Vol. 2. (3) Dogic, Z.; Fraden, S. Curr. Opin. Colloid Interface Sci. 2006, 11, 47. (4) Grelet, E. Phys. Rev. Lett. 2008, 100, 4. (5) Dogic, Z.; Fraden, S. Philos. Trans. R. Soc. London, Ser. A 2001, 359, 997. (6) Zhang, Z.; Krishna, N.; Lettinga, M. P.; Vermant, J.; Grelet, E. Langmuir 2009, 25, 2437. (7) Lettinga, M. P.; Grelet, E. Phys. Rev. Lett. 2007, 99, 197802. (8) Lettinga, M. P.; Barry, E.; Dogic, Z. Europhys. Lett. 2005, 71, 692. (9) Flynn, C. E.; Lee, S. W.; Peelle, B. R.; Belcher, A. M. Acta Mater. 2003, 51, 5867. (10) Nam, K. T.; Kim, D. W.; Yoo, P. J.; Chiang, C. Y.; Meethong, N.; Hammond, P. T.; Chiang, Y. M.; Belcher, A. M. Science 2006, 312, 885. (11) Dujardin, E.; Peet, C.; Stubbs, G.; Culver, J. N.; Mann, S. Nano Lett. 2003, 3, 413.

Langmuir 2010, 26(13), 10593–10599

obtain templates for the subsequent coating with inorganic materials.9-13 The fd virus is a rodlike virus with a length of 880 nm and a diameter of 6.6 nm.14 It is a natural bacteriophage that can be grown in male strains of Escherichia coli (here the XL1-blue strain). Each virus consists of a single-stranded circular DNA molecule packed in a cylindrical capsid of 2700 identical major coat proteins (p8) and a few copies each of four minor coat proteins that cap each end (Scheme 1A).15 Each p8 consists of 50 amino acid residues and can be divided into three parts starting from the N terminus: the hydrophilic solvent-exposed part (residues 1-20), the intermediate hydrophobic part (residues 21-39), and the basic part (residues 40-50). The basic part is buried inside the capsid, forming a complex with DNA by electrostatic balance,15 the hydrophobic part acts as the anchor for the self-assembly of p8 into the capsid, and the hydrophilic part determines the surface properties of fd. Two amino groups (residue Lys8 and the N terminus) and five acidic residues (Glu2, Asp4, Asp5, Asp12, and Glu20) are located within the solventexposed part (Scheme 1B), resulting in a negatively charged virus above its isoelectric point (IEP) of pH ∼4.216,17 and a net electrical charge density of about 3.4 e-/nm at pH 8.2. Below its IEP, fd is positively charged but the virus has limited stability at such low pH.17,18 Our goal is to obtain well-defined viruses that are permanently positively charged and are structurally stable over (12) Knez, M.; Sumser, M.; Bittner, A. M.; Wege, C.; Jeske, H.; Martin, T. P.; Kern, K. Adv. Funct. Mater. 2004, 14, 116. (13) Zhang, Z.; Buitenhuis, J. Small 2007, 3, 424. (14) Berkowitz, S. A.; Day, L. A. J. Mol. Biol. 1976, 102, 531. (15) Marvin, D. A. Curr. Opin. Struct. Biol. 1998, 8, 150. (16) Purdy, K. R.; Fraden, S. Phys. Rev. E 2004, 70, 061703. (17) Zimmermann, K.; Hagedorn, H.; Heuck, C. C.; Hinrichsen, M.; Ludwig, H. J. Biol. Chem. 1986, 261, 1653. (18) Glucksman, M. J.; Bhattacharjee, S.; Makowski, L. J. Mol. Biol. 1992, 226, 455.

Published on Web 04/30/2010

DOI: 10.1021/la100740e

10593

Article Scheme 1. Schematics of the Structure and Surface Modifications of the fd Virusa

a (A) (Left) Overview of fd. The yellow rodlike part is the capsid consisting of p8’s. On each end, there are a few copies of two minor coat proteins shown in green and pink. (Center) Bottom and side views of the ribbon structure of a part of the fd showing how the R-helices of p8 assemble along the virion axis in a helical way. (Right) Ribbon structure of a single p8 with the solvent-exposed part highlighted by the dashed rectangle. The stick at the N terminal is the nonhelical part consisting of five amino acid residues. The side groups of the residues are displayed in green. The side groups targeted in the current work are additionally highlighted where the blue parts are amino groups and the pink parts are acid groups. (B) Amino acid residue sequence of p8. The N terminus is to the left. Charged residues chemically accessible in the solvent-exposed part of p8 are indicated by the rectangular boxes; the wavy line indicates uncharged amino acid residues. (C) Chemical modifications described in this work. EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; NH2-R, amine compounds with at least one amino group; wavy line, poly(ethylene glycol).

a wide range of pH. To do so, it is necessary to change the ratio of the positive functional groups (amino groups) to the negative ones (the carboxyl groups) in the solvent-exposed part of coat protein p8. This can be done by transforming the overcrowded carboxyl residues either into the neutral state or even into the positively charged state. Alternatively, genetic modification can also be used to change the properties of the virus by the introduction of new amino acid residues onto the surface of the virus.19,20 Here we use a chemical modification of the carboxyl groups on the surface of the virus. For the chemical modification of carboxyl groups, carbodiimides are commonly used to convert them into reactive intermediate esters (O-acylisourea) that then react with a nucleophilic (19) Liu, A.; Abbineni, G.; Mao, C. Adv. Mater. 2009, 21, 1001. (20) Iannolo, G.; Minenkova, O.; Petruzzelli, R.; Cesareni, G. J. Mol. Biol. 1995, 248, 835. (21) Means, G. E.; Feeney, R. E. Bioconjugate Chem. 1990, 1, 2. (22) Strable, E.; Finn, M. G. In Viruses and Nanotechnology; Manchester, N., Steinmetz, N. F., Eds.; Current Topics in Microbiology and Immunology; SpringerVerlag: Berlin, 2009; Vol. 327, p 1.

10594 DOI: 10.1021/la100740e

Zhang et al.

reagent such as an amine or hydrazide, resulting in a stable amide bond.21-27 In this work, a water-soluble carbodiimide, 1-ethyl3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC), was used to activate the carboxyl groups of Glu2, Asp4, Asp5, Asp12, and Glu20. The resulting O-acylisourea then reacts with the amino compounds, including ethylenediamine (EDA), N,N-dimethylethylenediamine (DMEDA), and 1-(2-aminoethyl)piperazine (AEPA). These amino compounds have a positively charged group as well as a nucleophilic primary amino group that can react with the activated carboxyl group so that one positive charge is introduced while one negatively charged carboxyl group is transformed into a neutral amide bond. In this way, the surface charge of fd becomes positive over a wide pH range without losing its structural integrity. In addition, modifications of carboxyl groups were also combined with modifications of the surface amino groups. The surface amino groups from the lysine residue and the N terminus can be easily modified with several agents such as N-hydroxysuccinimide (NHS) esters, isothiocyanates, and aldehydes. Here, several organic compounds were tested to protect the endogenous amino groups from reacting with activated carboxyl groups, during which undesirable cross-linked viruses may be produced. It turns out that grafting the active NHS ester of poly(ethylene glycol) (PEG) to the amino groups is especially efficient for this purpose. The modifications described in this work result in modified viruses with intriguing properties. For example, one of the modified viruses forms a pure nematic phase that is different from the cholesteric phase of the unmodified wild-type viruses. Furthermore, a mixture of two kinds of PEG-grafted fd’s bearing opposite charges can flocculate reversibly, which can be controlled by tuning the salt concentration to change the balance between the steric interactions due to the grafted PEG layer and the electrostatic attractions due to the opposite charge. These modified viruses might be an interesting model system in soft condensed matter physics (e.g., in the study of polyelectrolyte complexes or liquid-crystalline phase behavior).

2. Experimental Section 2.1. Materials. Ethylenediamine dihydrochloride (EDA), N, N-dimethylethylenediamine (DMEDA), 1-(2-aminoethyl)piperazine (AEPA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC), and monomethoxy-terminated poly(ethylene glycol) succinimidyl propionate (mPEG-SPA, MW = 5 kDa, g80%) were purchased from Sigma-Aldrich and used as received. HCl (25%) and absolute ethanol (99.9%) were obtained from KMF Laborchemie Handels GmbH. Water from a Millipore Milli-Q10 system was always used. The fd virus was grown and purified following standard biochemical protocols using the XL1-blue strain of E. coli as the host bacteria (Supporting Information).28 The viruses will be referred to as wt-fd (i.e., wild-type fd virus), and care was taken to avoid contamination by amine compounds before using the virus in chemical modifications. For physical measurements, except as noted otherwise, the standard buffer is Tris-HCl buffer (20 mM Tris in water, pH adjusted to 8.2 by HCl), the ionic strength (I) of which was adjusted by adding various amounts of NaCl. (Note that 20 mM Tris at pH 8.2 corresponds to I = 10 mM.) (23) Nakajima, N.; Ikada, Y. Bioconjugate Chem. 1995, 6, 123. (24) Hoare, D. G.; Koshland, D. E. J. Biol. Chem. 1967, 242, 2447. (25) Gillitzer, E.; Willits, D.; Young, M.; Douglas, T. Chem. Commun. 2002, 2390. (26) Steinmetz, N. F.; Lomonossoff, G. P.; Evans, D. J. Langmuir 2006, 22, 3488. (27) Yacoby, I.; Bar, H.; Benhar, I. Antimicrob. Agents Chemother. 2007, 51, 2156. (28) Sambrook, J.; Russell, D. W. Molecular Cloning: A Laboratory Manual, 3rd ed.; Cold Spring Harbor Laboratory Press: New York, 2001.

Langmuir 2010, 26(13), 10593–10599

Zhang et al.

2.2. Modification of the Surface Amino Groups of wt-fd. To graft PEG to the amino groups of the coat proteins, the method in ref 5 was followed with some minor changes. mPEGSPA (80 mg) was added to 5 mL of a wt-fd dispersion (2 mg mL-1 viruses in 100 mM sodium phosphate buffer, pH 8.2), and the mixture was homogenized by gentle shaking. The grafting reaction was left standing for at least 12 h. To remove the residual mPEG-SPA, three cycles of centrifugation at 100 000g for about 6 h followed by redispersion of the sediment in buffer were performed. Hereafter, we refer to the resulting PEG-grafted fd as PEG-fd. A portion of purified PEG-fd was dispersed in TrisHCl buffer with I = 110 mM, and the concentration, CI-N, at which the isotropic-nematic phase transition occurs was then determined. CI-N ≈ 15 mg mL-1 (compared to 21 mg mL-1 for wt-fd under the same conditions) indicated successful PEG grafting.29

2.3. Modification of the Surface Carboxyl Groups of the fd Virus. Solutions of the organic amine (EDA, DMEDA, or AEPA) were first prepared and adjusted to a pH of about 5 with diluted HCl or NaOH before addition to the virus dispersion. The wt-fd or PEG-fd dispersion for the modification was prepared by diluting the stock virus dispersions with the organic amine solution. Then the pH of the final dispersion was measured, and if it changed by the addition of the fd dispersion, it was adjusted to 5-5.5 again. After that, excess EDAC was added with constant stirring. A typical procedure is as follows: the pH of a 200 mM EDA solution was adjusted to about 5. Then, 4.5 mL of this solution was mixed with 0.5 mL of a wt-fd stock dispersion of 20 mg mL-1, resulting in 5 mL of a wt-fd dispersion of 2 mg mL-1 in EDA aqueous solution. The pH of the resulting dispersion was adjusted to a value of between 5 and 5.5. To this dispersion, 0.4 mmol of EDAC was added. The reaction was allowed to proceed at room temperature for 5 h, and then the same amount of EDAC was added again. The final dispersion was dialyzed against Millipore water to remove residual amines and other byproducts. After dialysis, the viscous dispersions show permanent birefringence between crossed polarizers, a characteristic of the nematic liquid-crystalline phase. This observation was taken as the first indication of the stability of the viruses after modification. The dialyzed dispersions were further centrifuged at 12 000g to remove potential cross-linked fd viruses (Results and Discussion). 2.4. Electrophoresis. The electrophoretic mobility of modified fd and wt-fd was measured with a Zetasizer 2000 equipped with an aqueous dip cell (Malvern, England). All samples consisted of 0.1 mg mL-1 viruses in a 2 mM Tris-HCl buffer at pH 8 (I = 1 mM). An average electrophoretic mobility from 30 measurements was converted to an apparent zeta potential using the Smoluchowski formula (ξ = 4ημ/ε, where μ, η, and ε are the mobility of the particle, the viscosity, and the dielectric constant of the dispersion, respectively). 2.5. Determination of the Isoelectric Point (IEP). Following the electrophoresis measurement, some samples of wt-fd and modified viruses were titrated with dilute HCl or NaOH to different pH values. For each pH, electrophoresis measurements were performed to obtain the apparent zeta potentials, ξ. The pH at which the sign reversal of ξ occurs was taken as the IEP.

2.6. Flocculation Experiment of Charge-Reversed Viruses. The positive charge properties of the modified viruses were first checked by mixing comparable amounts of wt-fd and modified viruses in the Tris-HCl buffer or by mixing the modified viruses with the polyethyleneimine (PEI) polycation. Furthermore, the effect of mixing negatively charged and positively charged PEGfd was investigated in detail in order to demonstrate the subtle balance between electrostatic and steric interactions. For this purpose, equal portions of positively charged PEG-fd (obtained by the above modification with DMEDA) and negatively charged (29) Grelet, E.; Fraden, S. Phys. Rev. Lett. 2003, 90, 198302.

Langmuir 2010, 26(13), 10593–10599

Article PEG-fd were mixed to form dispersions with a total virus concentration of 0.5 mg mL-1 in a 2 mM Tris-HCl buffer (pH 8.2). Varying amounts of NaCl were added to change the ionic strength (I), and the occurrence of flocculation was observed with the naked eye. 2.7. SDS-PAGE. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) was performed following Laemmli’s method.30 Wild-type or modified fd viruses were denatured in a buffer containing 1.6% (w/v) SDS, 1% (v/v) β-mercaptoethanol, 12.5% (v/v) glycerol, 0.0025% (w/v) bromophenol blue, and 50 mM Tris (pH 7.6) (final concentration). The resulting mixture of p8, minor proteins, phage DNA, and other agents was loaded onto an 18% gel. Usually 1-3 μg of virus was used. Commercially available molecular-weight markers (Amersham Biosciences) were applied to at least one lane of each gel. Gels were stained with Coomassie Brilliant Blue R-250 in order to visualize the protein bands. p8 is the dominant protein and is intensely stained. 2.8. Liquid-Crystalline Behavior. When the concentration of the rodlike virus is above a certain threshold, a liquid-crystal (here nematic/cholesteric) phase will form, which is manifested by permanent birefringence. If a sample is too dilute, then only flow birefringence due to temporary alignment induced by flow might be observed. Both permanent birefringence and flow birefringence were checked by placing or shaking sample vials between crossed polarizers. Some samples of modified fd and wt-fd were placed into 1.5 mm quartz capillaries that were flame sealed and checked with polarized optical microscopy. 2.9. Static and Dynamic Light Scattering. Static and dynamic light scattering measurements were performed on an ALV/CGS-8F goniometer system with a 632.8 nm HeNe laser and an ALV-5000 multiple tau digital correlator (ALV, Germany). All measurements were carried out at 20 C. The modified fd or wild-type fd was dispersed in 20 mM Tris-HCl buffer (pH 8.20) at a concentration of 0.05 mg mL-1. To check for a possible concentration dependence, one sample of 0.025 mg mL-1 wt-fd (which is less than the overlapping concentration, i.e., 0.04 mg mL-1) was measured and no difference with the 0.05 mg mL-1 sample was found. All samples were centrifuged at 1800g for at least 12 h in order to remove dust. Static light scattering measurements were corrected for the scattering of the solvent. The scattering intensity was collected at different scattering angles ranging from 30 to 120. The autocorrelation functions were recorded at 40. This relatively small scattering angle was chosen to detect potential aggregates of viruses due to intervirus cross linking, if any. 2.10. Mass Spectrometry. MALDI-TOF mass spectrometry (MS) was performed to identify a series of modification products. The coat proteins were first denatured and separated into bands containing single or cross-linked proteins using SDSPAGE as described before. For the chemically modified virus, where multiple bands were observed, only the monomeric band located at a position similar to that in wt-fd was analyzed by MS. To prepare samples for MS, the excised and destained bands were digested either enzymatically or chemically using trypsin or CNBr, respectively. First, trypsine digestion was tried, but no clear peaks could be identified in the spectrum. Then CNBr cleavage was tested because it cuts coat protein p8 at only one point close to the middle of the hydrophobic part, thereby increasing the solubility and leaving all potential modification sites on one fragment with an easily measurable molecular weight. We followed the procedure of van Montfort et al.31 with a few minor modifications (further details in Supporting Information). Spectra were recorded on a Voyager-DE STR biospectrometry workstation (Applied Biosystems) and on an Ultraflex III (Bruker Daltonics). (30) Laemmli, U. K. Nature 1970, 227, 680. (31) van Montfort, B. A.; Doeven, M. K.; Canas, B.; Veenhoff, L. M.; Poolman, B.; Robillard, G. T. Biochim. Biophys. Acta 2002, 1555, 111.

DOI: 10.1021/la100740e

10595

Article

Zhang et al.

Table 1. Chemical Modification of Amino Groups and Carboxyl Groups of the fd Virus

a The electrophoretic mobility with the apparent zeta potential given in parentheses (Experimental Section).

3. Results and Discussion The different modifications are summarized in Table 1, showing combinations of modifications as well as single modifications of either amino or carboxyl groups. For modifications of the carboxyl groups for charge reversal, the positive electrophoretic zeta potentials clearly indicate a successful modification. All dispersions of modified fd show permanent birefringence after dialysis, which is a strong indication that the rodlike shape of the virus remained intact. In the following text, we will refer to the modified viruses with the nomenclature “modification-fd”. For example, EDA-fd refers to the fd virus modified with ethylenediamine. 3.1. Modification of Surface Carboxyl Groups with Amines. 3.1.1. Reaction Conditions. The modification of carboxyl groups with amines takes place in aqueous media in a narrow pH range of 5 to 6 with excess amines and carbodiimides. Under these conditions, the pH is a key factor in the reaction. At high pH, carbodiimide will hydrolyze very quickly into an inactive form,32 and at low pH, the carboxyl groups are in the protonated state, rendering them again inactive for the reaction. The excess amines were used to suppress intervirus cross linking, which can result in strongly flocculated dispersions. (See also the discussion below.) Indeed, control experiments that were performed at a low amine concentration resulted in strongly flocculated dispersions. This situation makes it impossible to control the positive charge density by simply varying the ratio of amines to carboxyl groups. Further means used to minimize the degree of cross linking will be discussed later. 3.1.2. SDS-PAGE Analysis. Denatured wt-fd and the modified viruses were subjected to SDS-PAGE analysis (Figure 1A). In the case of wt-fd, only a single band of major coat protein p8 was observed as expected. In contrast, all of the modified fd samples show many distinct bands (Figure 1A). Besides the band of the single monomeric (modified) p8 protein that appears at the same position as that of wt-fd, other bands of species with a molecular weight corresponding to a dimer, trimer, tetramer, and so forth of p8 are also observed. Species consisting of up to eight p8’s can be (32) Gilles, M. A.; Hudson, A. Q.; Borders, C. L. Anal. Biochem. 1990, 184, 244.

10596 DOI: 10.1021/la100740e

Figure 1. (A) SDS-PAGE analysis of the coat proteins of fd viruses modified with different organic primary amines: lane MW, molecular weight markers; lane 1, wt-fd; lane 2, fd modified with ethylenediamine (EDA-fd); lane 3, fd modified with N,N-dimethylethylenediamine (DMEDA-fd); lane 4, fd modified with 1-(2aminoethyl)piperazine (AEPA-fd); and lane 5, fd modified with both poly(ethylene glycol) and DMEDA. (B) MALDI-TOF MS analysis of the solvent-exposed part of the coat protein of wt-fd and DMEDA-fd as obtained after CNBr cleavage. For DMEDA-fd, only the monomeric coat protein band from SDS-PAGE (A) was analyzed. The typical peaks corresponding to expected fragments for DMEDA-fd are highlighted by arrows. (See the Supporting Information for details.) (C) Apparent zeta potential versus pH. The values shown are the isoelectric points (IEPs).

resolved with the 18% gel used in this work that is sensitive to proteins with a low molecular weight. Species with more than eight p8’s merge with each other and manifest themselves as a broad band (lanes 2 and 4 in Figure 1A). The species with a lower number of p8’s are dominant, as estimated from the total intensity of the bands. It is quite obvious that these species containing two or more p8’s should originate from the chemical cross linking of p8. After the activation step of the carboxyl group, any species with an amino group (or other nucleophilic groups) can attack the active intermediate ester (O-acylisourea) to form an amide (or other) bond.33 Apart from the attack by the added amines that are dominant in the reaction system, the amino group of the Lys8 residue and the N terminus of coat protein p8 can also react with the activated carboxyl groups. Obviously, these latter reactions may result in three situations. First, an amino group may react with the neighboring carboxyl groups in the same p8, resulting in a modified p8 with a cyclic structure (intraprotein cross linking). If this reaction occurs, then it is expected that the modified p8 will appear in the same band as the intact p8 in SDS-PAGE because (33) Williams, A.; Ibrahim, I. T. Chem. Rev. 1981, 81, 589.

Langmuir 2010, 26(13), 10593–10599

Zhang et al.

there is no dramatic change in molecular weight. In the second case, amino groups of one p8 coat protein may react with the activated carboxyl groups of the neighboring p8 of the same virus, resulting in cross-linked p8 (intravirus cross linking). Because of the multireactive sites of each p8, one can expect that intravirus cross-linking produces different kinds of cross-linked species with a varying number of p8’s and a molecular weight that is several times that of a single p8. The third possible reaction is that amino groups of one virus react with activated carboxyl groups of another virus so that two or more viruses will be chemically cross linked together (intervirus cross linking). It is expected that intraprotein and intravirus cross linking is much more frequent than intervirus cross linking, in view of the proximity of the amino and carboxyl groups within one virus or even one protein (Scheme 1A). This will be confirmed in the case of fd modified with poly(ethylene glycol) and also by light-scattering measurements on wild type and modified fd (as will be discussed later). Note that there is no band corresponding to any oligomer containing more than eight p8’s in the case of DMEDA-fd, in contrast to EDA-fd or AEPA-fd (Figure 1A). This implies that the degree of cross linking for DMEDA-fd is much lower than that for EDA-fd or AEPA-fd. This is understandable because EDA and AEPA have two active amino groups (two primary groups in the case of EDA and one primary group and one secondary amino group in AEPA), both of which can react with the activated carboxyl groups and result in cross linking. In contrast, DMEDA has one primary amino group and one inactive tertiary amino group, so the cross linking in the case of DMEDA can be attributed only to the endogenous amino groups of fd. In this regard, DMEDA is a good candidate for the modification of carboxyl groups. 3.1.3. Mass Spectrometry. MALDI-TOF mass spectrometry was used to identify the modifications (Figure 1B). In the case of modified viruses, only the monomeric protein band at the same position of SDS-PAGE as for wild-type fd was used in the CNBr cleavage procedure as described in the Experimental Section. For fd modified with EDA and DMEDA, peaks with the expected shifts in molecular weight can be identified that correspond to two, three, or four modified carboxyl groups per coat protein, with or without one intraprotein cross link as discussed previously. As expected, the peak without any modification as observed in wt-fd is no longer found in the spectra of EDA-fd and DMEDA-fd. Surprisingly, no peak corresponding to the modification of all five carboxyl groups is found. Still, if we assume an average degree of modification of about three carboxyl groups per coat protein, then this can qualitatively explain the result from electrophoresis because the modified fd then has five positively charged groups and two negatively charged groups per coat protein, as compared to five negatively and two positively charged groups per coat protein for wt-fd. A more detailed discussion of the mass spectrometry analysis can be found in the Supporting Information. 3.1.4. Electrostatic Properties. Electrophoresis measurements reveal that all of the modified viruses possess positive charges. The viruses modified with three kinds of amines possess an almost equal electrophoretic zeta potential at pH 8 and an ionic strength of about 1 mM (Table 1, entries 1-3). This indicates a comparable degree of modification for all three cases. Similar to other polyelectrolytes, flocs form quickly when mixing negatively charged wt-fd and the modified viruses. Also, polycations such as poly(ethyleneimine) can easily flocculate wt-fd, but they have no effect on fd modified with the above organic amines. These simple qualitative experiments further confirm the positive charge of the modified viruses. Langmuir 2010, 26(13), 10593–10599

Article

For EDA-fd, the isoelectric point (IEP) was found to shift from pH 4.2 for wt-fd to pH 10.5, close to the pKa of primary amines in proteins (Figure 1C). Above this pH, the modified viruses will evolve into negatively charged states. As revealed by mass spectrometry, there are still some unmodified carboxyl groups left on each coat protein that offer negative charges above the IEP. Nevertheless, the viruses are demonstrated to be overall positively charged over a wide range of pH. 3.2. Modification of Surface Amino Groups to Minimize Intervirus Cross Linking. As discussed before, intervirus cross linking will lead two or more viruses that cross link together permanently as indicated by the increasing turbidity during the modification. This can be alleviated by using a large excess of amine and choosing amine reagents with only one active amino group such as DMEDA. Centrifugation at 12 000g is also an efficient way to remove the cross-linked viruses. Performing the modification at very low fd concentration might be another option but is not practical from an application point of view. Taking into account the dual-functional nature of fd with both amino and carboxyl groups on its surface, protection of the endogenous amino groups is obviously a reasonable solution. Several common reagents were tested to block the endogenous primary amino groups of the fd virus, of which methyl acetimidate (MA)34 results in an almost complete blocking of the amino groups. The promising combination of MA blocking of the amino groups with DMEDA modification of the carboxyl groups of the viruses leads to charge-reversed viruses almost free of cross links as revealed by SDS-PAGE analysis (results not shown here). However, the modified virus dispersions became unstable and flocculated slowly for unknown reasons. Therefore, complete blocking of the endogenous amino groups of the fd virus was not further pursued, and instead we turned to steric hindrance offered by a grafted polymer layer. For this, poly(ethylene glycol) (PEG) was grafted to the amino groups on fd (referred to as PEGfd) to protect two viruses from approaching each other. In this way, intervirus cross linking is expected to be diminished, which was qualitatively evidenced by the fact that clear dispersions were obtained after PEG-fd was subjected to the modification with EDA or DMEDA. Nevertheless, SDS-PAGE analysis of PEG-fd modified with DMEDA gives a multiple band pattern very similar to that of wt-fd modified with DMEDA (lane 5 in Figure 1A), which indicates that the intervirus cross linking is only a small fraction of the total cross linking. Because only around 400 out of 5400 of the surface amino groups will be coupled with PEG,29 the rest can still take part in intraprotein and intravirus cross linking although intervirus cross linking is expected to decrease to a very low degree because of the grafted PEG layer. 3.3. Static and Dynamic Light Scattering. Static and dynamic light scattering measurements were performed to check for intervirus cross linking as discussed above. Figure 2A shows the static light scattering (SLS) of wt-fd and a series of modified fd’s as given in the Figure legend. In addition, a calculated scattering curve35 for rotationally averaged, thin rods of 880 nm length is shown for comparison. The diameter of the rods, with or without a PEG grafting layer, is very small compared to the wavelength of light and is therefore not important to the shape of the static light scattering curves. The small difference between the calculated curve and the different measured curves might be attributed to small amounts of residual dust, differences in flexibility of the (modified) fd viruses, and small deviations from (34) Armstrong, J.; Hewitt, J. A.; Perham, R. N. EMBO J. 1983, 2, 1641. (35) Buitenhuis, J.; Dhont, J. K. G.; Lekkerkerker, H. N. W. J. Colloid Interface Sci. 1994, 162, 19.

DOI: 10.1021/la100740e

10597

Article

Zhang et al.

Figure 3. Flocculation experiments of oppositely charged PEGfd. (A) Schematic representation of fd grafted with PEG. (B) Typical experimental results. The NaCl concentration in the left vial is 10 mM, and it is 20 mM in the right one. Only in the left vial are flocculation and gelation observed. The gel is found on the wall above the dispersion, which is not visible. (C) Mechanism of the reversible flocculation of two kinds of PEG-fd bearing opposite charges. I represents the ionic strength.

Figure 2. Light scattering of wt-fd and modified fd. (A) SLS intensity at different scattering vectors. The solid line is the calculated scattering curve for rotationally averaged infinitely thin rods with a length of 880 nm in the Rayleigh-Gans-Debye approximation.35 The measured intensity is normalized to the calculated value at the highest measured angle. (B) Auto correlation functions from DLS at a scattering angle of 40. The curves are normalized to 1 at the lowest τ. Legend: () wild-type fd; (4) fd grafted with PEG; (O) fd modified with ethylenediamine (EDA); (0) fd modified with N,N-dimethylethylenediamine (DMEDA); and ()) fd grafted with PEG and then modified with N,N-dimethylethylenediamine (DMEDA).

the Rayleigh-Gans-Debye approximation as used for the calculated curve. Even for fd modified with EDA, where the highest degree of coat protein cross linking was found by SDS-PAGE, the deviation from the measurement for wt-fd is very small. Figure 2B gives the results of the dynamic light scattering (DLS) measurements at a scattering angle of 40 for a series of samples. As shown in Figure 2B, the renormalized autocorrelation functions for all of the samples almost superimpose onto each other. Small differences are discernible at the longer relaxation time without a clear correlation with an expected degree of aggregation. For example, the system with the longest relaxation time is DMEDA-PEG-fd, which is not expected to have any significant degree of intervirus cross linking because of the PEG grafting. Furthermore, EDA-fd is expected to be most sensitive to intervirus cross linking; nevertheless, the dynamic light scattering result for EDA-fd is very close to the result for wt-fd. Therefore, it seems that the small differences shown in Figure 2B might be due to an increase in the effective diameter after PEG grafting or (36) Maeda, T.; Fujime, S. Macromolecules 1985, 18, 2430.

10598 DOI: 10.1021/la100740e

differences in the flexibility of the (modified) fd viruses.36 In summary, SLS and DLS indicate that there is no or only a low degree of intervirus cross linking. 3.4. Reversible Flocculation of Oppositely Charged PEGfd. A subtle balance between the steric repulsion and electrostatic attraction was demonstrated by mixing a positively and a negatively charged PEG-fd with different salt concentrations (Figure 3). When the total ionic strength (I) is lower than 20 mM, visible flocs appear after mixing oppositely charged PEGfd. Besides the white flocs, some transparent gel also forms on the wall of the container above the surface of the liquid. However, no flocs or gel are observed at I > 20 mM, and the resulting mixtures stay clear (Figure 3B). This behavior can be compared to the work of Dogic and co-workers on the isotropic-nematic phase transition (I-N) of wild-type fd grafted with PEG with a molecular weight of 5K (PEG5K-fd). They found that above an ionic strength of about 20 mM the I-N transition of PEG5K-fd starts to deviate from that of wt-fd and essentially becomes independent of the ionic strength.5 The coincidence of the critical ionic strength for the I-N phase transition of PEG-fd and the flocculation of oppositely charged PEG-fd can be qualitatively understood by the following mechanism. At high ionic strength, the steric interaction dominates because the electrical double layer will be confined within the PEG layer and the behavior of the viruses becomes independent of the ionic strength or charge, whereas at low ionic strength the electrostatic interactions dominate (Figure 3). Therefore, at an ionic strength of 20 mM or higher, a mixture of positively and negatively charged PEG-fd behaves similarly to a dispersion of PEG-fd with the same surface charge. At low ionic strength, however, where the double layer extends beyond the PEG layer, a mixture of oppositely charged PEG-fd will form aggregates or a gel. On the basis of this explanation, it is expected that the flocculation or gelation is reversible. This was confirmed by the disappearance of flocculation and gelation in the original flocculated mixture when the NaCl concentration was increased to above 20 mM. Langmuir 2010, 26(13), 10593–10599

Zhang et al.

Article

other viruses of similar structure such as Pf1 do not form the same phase. The dramatic difference between EDA-fd and wt-fd stimulated our strong interest in the influence of surface modifications on the nematic phase of the rodlike virus, and detailed investigations will be presented elsewhere. We speculate that intraprotein cross linking and intravirus cross linking during the modifications might influence the interactions of coat proteins and DNA,39 the charge distribution pattern,38 and the flexibility or conformation of the virus, all of which might contribute to the formation of a nonchiral nematic phase for EDA-fd. Although the precise origin of the different behavior is not clear, it is demonstrated here that the chemical modifications represent an interesting way to tune the liquid-crystalline phase behavior of fd virus dispersions.

4. Conclusions

Figure 4. Polarization microscopy images of the liquid-crystalline phase of the wild-type and modified fd virus. The scale bar is 200 μm. (A) Homogeneous striplike fingerprints of a typical cholesteric phase of wild-type fd. C=49.6 mg mL-1 and I=15 mM. (B) Nematic phase observed in the case of fd modified with EDA. C=48 mg mL-1 and I = 15 mM.

3.5. Liquid-Crystalline Phase Behavior. All dispersions of modified fd show permanent birefringence above a certain concentration, demonstrating the formation of a liquid-crystalline phase. The nature of the liquid-crystalline phase of EDA-fd was checked by polarization microscopy and compared to that of wtfd (Figure 4). Surprisingly, only a pure, nonchiral nematic phase was found for EDA-fd, contrary to the nematic phase found for wt-fd, which is actually a cholesteric phase (i.e., chiral, with the nematic director rotating helically along an axis).37 There are still debates about the origin of this cholesteric phase29,38-40 because (37) Dogic, Z.; Fraden, S. Langmuir 2000, 16, 7820. (38) Kohlstedt, K. L.; Solis, F. J.; Vernizzi, G.; Olvera de la Cruz, M. Phys. Rev. Lett. 2007, 99, 30602.

Langmuir 2010, 26(13), 10593–10599

Charge-reversed fd viruses (i.e., positively charged fd within a wide range of pH) have been prepared by the chemical modification of the carboxyl groups on the surface of the virus by carbodiimide chemistry. All of the modification procedures in this work were performed under mild conditions that are benign to the fd virus. As expected, adding dispersions of positively and negatively charged fd together results in flocculation. This charge reversal can be combined with a steric stabilization layer of grafted poly(ethylene glycol) so that the above-mentioned flocculation can be made adjustable and reversible by changing the ionic strength of the dispersion. Furthermore, other properties that are not directly related to the surface charge also can be tuned, as was shown for EDA-fd, which forms a pure, nonchiral nematic phase in contrast to the cholesteric phase found for wt-fd. Altogether, these modifications result in fd viruses with new surface properties, which might be of interest as a model system for fundamental studies in soft condensed matter physics, for example, for studies on polyelectrolyte effects and liquid-crystalline phase behavior. Acknowledgment. We thank Pavlik Lettinga for stimulating discussions and Eric Grelet from CRPP for assistance with the characterization of the liquid-crystalline phase. Z.Z. also thanks Prof. Jan Vermant (K.U. Leuven) for his generous support during the preparation of this manuscript through EU-funded Nanodirect FP7-NMP-2007-SMALL-1 project 213948. Supporting Information Available: Details of the MALDITOF mass spectrometry analysis, transmission electron microscopy results, and the preparation of the fd virus. This material is available free of charge via the Internet at http:// pubs.acs.org. (39) Tomar, S.; Green, M. M.; Day, L. A. J. Am. Chem. Soc. 2007, 129, 3367. (40) Tombolato, F.; Ferrarini, A.; Grelet, E. Phys. Rev. Lett. 2006, 96, 258302.

DOI: 10.1021/la100740e

10599