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Cowpea Mosaic Virus for Material Fabrication: Addressable Carboxylate Groups on a Programmable Nanoscaffold Nicole F. Steinmetz, George P. Lomonossoff, and David J. Evans* Department of Biological Chemistry, John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom ReceiVed January 9, 2006. In Final Form: February 22, 2006 For the first time, decoration of surface-exposed carboxylate groups on Cowpea mosaic Virus particles is reported, thus increasing the number and types of addressable surface groups on this nanoscaffold. First, the addressabilty of carboxylates was demonstrated using a carboxylate-selective fluorescent dye, N-cyclohexyl-N′-(4-(dimethylamino)naphthyl)carbodiimide. Second, it was shown that the virions can be decorated with approximately 180 redox active, methyl(aminopropyl)viologen moieties by coupling to the surface carboxylates. The display of multiple redox centers on the virus particle surface may lead to the development of novel electron-transfer mediators in redox catalysis, to biosensors, and to nanoelectronic devices such as molecular batteries.
Introduction The use of biomolecules for material fabrication has become popular during recent years;1-3 especially, plant viral capsids have received particular attention.4,5 Plant viruses, such as Cowpea mosaic Virus (CPMV), display a number of features that can be exploited for nanoscale biomaterial fabrication. The major advantage of viral capsids is their discrete size and geometry with a high degree of symmetry and polyvalency. In addition, the genetic, biological, and physical properties of CPMV are well defined.6,7 Plant viruses are noninfectious toward other organisms and present no biological hazard. Also, inoculation and purification are simple and quick. CPMV infects legumes, and high expression levels can be reached within a short period of time; yields on a gram scale can be obtained from 1 kg of infected leaf material. CPMV particles are extremely robust: they maintain their integrity at 60 °C (pH 7) for at least 1 h and at pH values from 3.5 to 9.0 indefinitely at room temperature. Furthermore, the native virus can tolerate organic solvents such as dimethyl sulfoxide (DMSO) at concentrations up to 50% for at least several hours.8 These properties facilitate chemical functionalization of the capsid surface. The CPMV viral capsid has a diameter of approximately 28 nm, the structure of which is known to near atomic resolution.6 The virions are formed by 60 copies of two different types of coat proteins; the small (S) and large (L) subunits are arranged with icosahedral symmetry. The S subunit folds into one domain (24 kDa), whereas the L subunit is comprised of two domains (41 kDa). The three domains form the asymmetric unit and are arranged in a pseudo T ) 3 surface lattice. Because of its topology, the virion can be regarded as a large dendrimer; the multiple copies of the asymmetric unit provide regularly spaced attachment units, which makes them a useful nanoscaffold, facilitating the attachment and presentation of different moieties on both the interior and exterior surfaces. In previous studies, both wild-type CPMV and CPMV mutants have been functionalized and decorated with a variety of * Corresponding author. E-mail:
[email protected]. (1) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128-4158. (2) Bittner, A. M. Naturwissenschaften 2005, 92, 51-64. (3) Ball, P. Nanotechnology 2005, 16, R1-R8. (4) Flynn, C. E.; Lee, S.-W.; Peelle, B. R.; Belcher, A. M. Acta Mater. 2003, 51, 5867-5880. (5) Arora, P. S.; Kirshenbaum, K. Chem. Biol. 2004, 11, 418-420. (6) Lin, T.; Chen, Z.; Usha, R.; Stauffacher, C. V.; Dai, J. B.; Schmidt, T.; Johnson, J. E. Virology 1999, 265, 20-34. (7) Lomonossoff, G. P.; Johnson, J. E. Prog. Biophys. Mol. Biol. 1991, 55, 107-137. (8) Wang, Q.; Lin, T.; Tang, L.; Johnson, J. E.; Finn, M. G. Angew. Chem., Int. Ed. 2002, 41, 459-462.
molecules,8-23 demonstrating the feasibility of utilizing CPMV as a molecular assembly in nanotechnology. Unique lysine9,15 and tyrosine residues21 on the external surface of wild-type virions have been selectively decorated by functional groups. In this paper, we describe, for the first time, the functionalization of carboxylate groups derived from aspartic and glutamic acids on the solvent-exposed surface of wild-type CPMV. This extends the number and type of accessible surface groups that allow covalent decoration by functional chemicals. Experimental Section The accessible surface profile of CPMV (the atomic coordinates for CPMV are available at http://viperdb.scripps.edu) indicates that there are seven solvent-exposed carboxylate groups on the particle surface: Asp 26, Asp 44, Asp 45, Asp 85, and Glu 135 on the S subunit, and Asp 273 and Glu 319 in the B domain of the L subunit. An additional carboxylate is derived from Glu 198, which is located in the solvent-exposed carboxy-terminus of the S subunit. However, because of the carboxy-terminal cleavage of 24 amino acids of the S subunit in the plant,24 the Glu 198 is only present in a small proportion of the particles. Furthermore, the carboxy-terminus of the S subunit, whether cleaved or not, is itself an exposed carboxylate. To probe the reactivity of the surface-exposed aspartic and glutamic acids and to achieve decoration of the viral nano-building-block, we have used a fluorescent carboxylate-selective chemical dye (Figure (9) Chatterji, A.; Ochoa, W. F.; Paine, M.; Ratna, B. R.; Johnson, J. E.; Lin, T. Chem. Biol. 2004, 11, 855-863. (10) Chatterji, A.; Ochoa, W. F.; Ueno, T.; Lin, T.; Johnson, J. E. Nano Lett. 2005, 5, 597-602. (11) Chatterji, A.; Ochoa, W.; Shamieh, L.; Salakian, S. P.; Wong, S. M.; Clinton, G.; Ghosh, P.; Lin, T.; Johnson, J. E. Bioconjugate Chem. 2004, 15, 807-813. (12) Blum, A. S.; Soto, C. M.; Wilson, C. D.; Brower, T. L.; Pollack, S. K.; Schull, T. L.; Chatterji, A.; Lin, T.; Johnson, J. E.; Amsinck, C.; Franzon, P.; Shashidhar, R.; Ratna, B. R. Small 2005, 1, 702-706. (13) Blum, A. S.; Soto, C. M.; Wilson, C. D.; Cole, J. D.; Kim, M.; Gnade, B.; Chatterji, A.; Ochoa, W. F.; Lin, T. W.; Johnson, J. E.; Ratna, B. R. Nano Lett. 2004, 4, 867-870. (14) Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.; Sharpless, K. B.; Finn, M. G. J. Am. Chem. Soc. 2003, 125, 3192-3193. (15) Wang, Q.; Kaltgrad, E.; Lin, T.; Johnson, J. E.; Finn, M. G. Chem. Biol. 2002, 9, 805-811. (16) Wang, Q.; Lin, T.; Johnson, J. E.; Finn, M. G. Chem. Biol. 2002, 9, 813-819. (17) Strable, E.; Johnson, J. E.; Finn, M. G. Nano Lett. 2004, 4, 1385-1389. (18) Raja, K. S.; Wang, Q.; Gonzalez, M. J.; Manchester, M.; Johnson, J. E.; Finn, M. G. Biomacromolecules 2003, 4, 472-476. (19) Raja, K. S.; Wang, Q.; Finn, M. G. ChemBioChem 2003, 4, 1348-1351. (20) Medintz, I. L.; Sapsford, K. E.; Konnert, J. H.; Chatterji, A.; Lin, T.; Johnson, J. E.; Mattoussi, H. Langmuir 2005, 21, 5501-5510. (21) Meunier, S.; Strable, E.; Finn, M. G. Chem. Biol. 2004, 11, 319-326. (22) Steinmetz, N. F.; Lomonossoff, G. P.; Evans, D. J. Small, in press. (23) Portney, N. G.; Singh, K.; Chaudhary, S.; Destito, G.; Schneemann, A.; Manchester, M.; Ozkan, M. Langmuir 2005, 21, 2098-2103.
10.1021/la060078e CCC: $33.50 © 2006 American Chemical Society Published on Web 03/09/2006
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Figure 1. Schematic of (a) the NCD4-labeled CPMV capsid, CPMV[NCD4]n, and (b) the MAV-decorated CPMV nanoscaffold, CPMV[MAV]n. 1a). Furthermore, we have utilized the carboxylate groups as attachment points for an organic, redox-active viologen derivative, methyl(aminopropyl)viologen (MAV; Figure 1b). The propagation and purification of the viruses were performed by standard protocols,25 and the purified virions were stored at 4 °C in 10 mM sodium phosphate buffer pH 7.0. The concentration of purified virions was determined by photometrical measurements (the molar extinction coefficient of CPMV at a wavelength of λ ) 260 nm is ) 8.1 mL mg-1 cm-1) or by Bradford assay. Chemical derivatization of the virions with the carboxylateselective fluorescent dye N-cyclohexyl-N′-(4-(dimethylamino)naphthyl)carbodiimide (NCD4, Molecular Probes) was achieved by shaking the CPMV virus (4 mg mL-1) with an 1800-fold excess (to ensure maximum decoration) of dye in a buffered solution containing 20 vol % DMSO. After incubation overnight, at 4 °C in the dark, the reaction mixture was purified with 100 kDa cutoff columns (Millipore). The columns were washed several times to make sure that all nonreacted dye was removed. The modified virions were resuspended in water, and the recovered yield of CPMV-[NCD4]n virions was approximately 70%. MAV [N-methyl-N′-(3-aminopropyl)-4,4′-bipyridinium diiodide] was synthesized according to published procedures in a two-step process. 1-methyl-4,4′-bipyridinium diiodide was synthesized as described by Kelly et al.26 and characterized by 1H NMR in d6DMSO: 9.13 ppm (d, 2H), 8.87 ppm (d, 2H), 8.65 ppm (d, 2H), 8.04 ppm (d, 2H), and 4.37 ppm (s, 3H). This was then converted to MAV27 and characterized by 1H NMR in d6-DMSO: 9.40 ppm (d, 2H), 9.30 ppm (d, 2H), 8.85 ppm (d, 2H), 8.77 ppm (d, 2H), 7.75 ppm (b, 1H), 4.79 ppm (t, 2H), 4.44 ppm (s, 3H), 2.91 ppm (t, 2H), and 2.29 ppm (quintet, 2H). The covalent coupling of MAV to CPMV capsids was achieved by a standard protocol for the formation of peptide bonds: N-ethylN′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, Novabiochem) was dissolved in water and used in a 1200 molar excess, and N-hydroxysuccinimide (NHS, Fluka) was dissolved in DMSO and used in a 7200 molar excess. Both reagents were used to activate the carboxylic acids and to accomplish the facile coupling of MAV to the virions. MAV was used in an 1800-fold molar excess to CPMV (to ensure maximum decoration). The final DMSO concentration of the reaction mixture was adjusted to 20 vol %, and the virus concentration was 4 mg mL-1. After shaking overnight at room temperature, hydroxylamine hydrochloride (ACROS Organics) (24) Taylor, K. M.; Spall, V. E.; Butler, P. J.; Lomonossoff, G. P. Virology 1999, 255, 129-137. (25) Wellink, J. Methods Mol. Biol. 1998, 81, 205-209. (26) Kelly, L. A.; Rodgers, M. A. J. J. Phys. Chem. 1994, 98, 6386-6391. (27) National Textile Center Annual Report, NTC Project C02-GT09; National Textile Center: Spring House, PA, 2004.
Langmuir, Vol. 22, No. 8, 2006 3489 in a molar ratio of 1:1 to EDC was added to deactivate undecorated carboxylates so as to minimize interparticle cross-linking. Aggregation via interparticle linkage can occur by the formation of peptide bonds between activated carboxylates and lysine residues on the CPMV surface. Chatterji et al.9 showed that wild-type CPMV displays five solvent-exposed lysine residues on its outer surface, which are all reactive, but to different degrees. To assess interparticle linkage, a control reaction using CPMV, EDC, and NHS was performed using conditions similar to those described above. No aggregation occurred, as shown by transmission electron microscopy (TEM), agarose gel electrophoresis, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (data not shown). Purification of the MAV-decorated virions was as follows: the precipitate that formed was removed by centrifugation (13000g, 10 min, room temperature); 100 kDa cutoff columns were used for purification of the derivatized virus particles; and the columns were washed until MAV was no longer detectable in the flow-through. The modified virions were resuspended in water, and the recovered yield of CPMV-[MAV]n virions was approximately 70%.
Results and Discussion The integrity of the CPMV-[NCD4]n and CPMV-[MAV]n virions, after derivatization, was confirmed by TEM (data not shown). In both cases, the particles were monodisperse with a diameter of approximately 30 nm. The dye-labeled CPMV[NCD4]n particles were characterized by UV/visible spectroscopy in 50% DMSO/H2O mixtures. NCD4 has two absorption maxima, one at 345 nm and another at ∼260 nm. The peak at 345 nm is well separated from the CPMV peak, which lies at 260 nm, and can therefore be used as an indicative peak to monitor covalent binding. The UV/visible spectra of NCD4, CPMV, and CPMV[NCD4]n shown in Figure 2 are consistent with the successful decoration of carboxylates with the dye. Because of the instability of the dye in aqueous solvent, a reliable extinction coefficient could not be obtained, preventing quantification of the number of bound dye molecules. Agarose gel electrophoresis of intact virions further supported successful labeling with NCD4 and covalent decoration with MAV (Figure 3a). Virions (10 µg) were analyzed (added loading dye, MBI Fermentas, Inc.) on a 1.2% agarose gel. The net negative charge of the CPMV capsid causes migration in the electric field toward the anode. Detection of the viral particles can be achieved by either ethidium bromide staining of the encapsidated RNA and visualization under UV light or Coomassie staining of the viral capsid protein (data not shown). Intact virions of CPMV can be separated into two forms: a higher and lower mobility form, according to their migration toward the anode in the electric field. These forms are derived from the slow (s) and fast (f) forms of the S subunit. The conversion from the s to the f form is a consequence of a carboxy-terminal cleavage of 24 amino acids of the S subunit.24 The higher mobility band in the agarose gel represents virions with a cleaved S, whereas the slower mobility band represents virions with a full-length S. The CPMV-[NCD4]n particles in agarose gels were also visualized by UV light with and without ethidium bromide staining. The fluorescence of the particles on agarose gel without ethidium bromide staining confirms that the virions have been labeled with NCD4. After ethidium bromide staining, a comparison to native CPMV particles showed that the dye does not affect the mobility in the gel. In contrast, MAV-derivatized CPMV particles are strongly retarded in their mobility. The retardation can be explained by a charge effect. The high number of positive charges associated with the viologen moieties will slow the migration of the modified proteins toward the anode. The dye-labeled CPMV-[NCD4]n virions were further characterized by denaturating SDS-PAGE using a 12% bis-tris gel.28 (28) Laemmli, U. K. Nature 1970, 227, 680-685.
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Figure 2. UV/visible spectra of (a) NCD4, (b) CPMV particles, and (c) CPMV-[NCD4]n (CPMV particles after derivatization with the carboxylate-specific fluorescent dye). The peak at 345 nm of CPMV-[NCD4]n indicates that the carboxylate groups are addressable.
Figure 3. (a) CPMV particles on a 1.2% agarose gel with and without ethidium bromide (EtBr) staining. Shown are native CPMV virions, CPMV-[NCD4]n, and CPMV-[MAV]n particles. (b) Wildtype CPMV particles and CPMV-[NCD4]n virions after separation on a denaturating 12% SDS gel. Detection of the viral capsid subunitssthe S and L subunitsswas achieved by Coomassie staining (left) or by visualization of the modified fluorescent CPMV-[NCD4]n proteins under UV light (right).
This showed that the dye was attached to both the S (s and f form) and the L subunits (Figure 3b), which is an expected observation since solvent-exposed aspartic and glutamic acid residues are present on both subunits. Electrochemical studies on CPMV-[MAV]n showed them to be redox active nanoparticles. Experiments were performed in a three-compartment glass cell with a glassy carbon disk working electrode, a glassy carbon counter electrode, and a Ag/AgCl reference electrode in 0.1 M sodium phosphate buffer (pH 7.0) under an inert atmosphere of argon. Cyclic voltammetric studies on viologen-decorated CPMV-[MAV]n nanoparticles showed the characteristic two successive, one-electron, reversible steps of the methyl viologen moieties. The two reduction potentials, E0/ ) -0.65 and -0.97 V versus the Ag/AgCl electrode for the virus-bound viologens, were indistinguishable from those of methyl viologen in solution (Figure 4a). A large difference in E0/ is not expected, as the methyl viologen-N-propylamine group and methyl viologen-N-propylamide groups will have similar inductive properties due to the intervening propyl group. Therefore, the behavior of attached viologen moieties, from an electrochemical point of view, is similar to that of viologen in solution. Evidently, the attached moieties behave as independent, electronically isolated units. Peak currents were measured, and the linear plot of ip versus υ1/2 (R ) 0.996) showed that the reduction was controlled by a diffusion process (Figure 4b). The number of viologen molecules attached to each CPMV virion can be estimated by use of the Randles-Sevcik equation29 to be 180 ( 20. Thus, around three carboxylates per asymmetric unit (29) Adams, R. N. Electrochemistry at Solid Electrodes; Marcel Dekker: New York, 1969.
Figure 4. (a) Cyclic voltammogram of CPMV-[MAV]n at a scan rate of 150 mV and (b) a linear plot of the measured current versus the square root of the scan rate.
of the virion were covalently functionalized by the viologen derivative, corresponding to at least 30% of the carboxylates.
Conclusions In conclusion, we have shown here, for the first time, that solvent-exposed carboxylates, arising from aspartic and glutamic acids on the outer surface of CPMV, can be modified with chemical moieties. Carboxylate groups on both the S and L subunits were bound by a carboxylate-selective dye. The virions can also be decorated with approximately 180 organic, redoxactive molecules, which are essentially noninteracting. Therefore, the system could act as a multielectron reservoir, which may lead to the development of nanoscale electron-transfer mediators in redox catalysis, molecular recognition, and amperometric biosensors, and to nanoelectronic devices such as molecular batteries. Acknowledgment. The Biotechnology and Biological Sciences Research Council, UK, and the EU, Marie Curie Early Stage Training Scheme, MEST-CT-2004-504273 (N. F. S.) are acknowledged for funding. Christopher J. Pickett is thanked for helpful discussions, and Saad K. Ibrahim is thanked for assistance with the electrochemistry studies. Supporting Information Available: Details for the calculation of the number of viologen moieties presented on CPMV employing the Randles-Sevcik equation are given. This material is available free of charge via the Internet at http://pubs.acs.org. LA060078E
