Disulfide Bond: Dramatically Enhanced Assembly Capability and

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Disulfide Bond: Dramatically Enhanced Assembly Capability and Structural Stability of Tobacco Mosaic Virus Nanorods Kun Zhou, Feng Li, Gaole Dai, Chun Meng, and Qiangbin Wang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm400445m • Publication Date (Web): 13 Jun 2013 Downloaded from http://pubs.acs.org on June 15, 2013

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Disulfide Bond: Dramatically Enhanced Assembly Capability and Structural Stability of Tobacco Mosaic Virus Nanorods Kun Zhou†‡, Feng Li†, Gaole Dai†‡, Chun Meng‡, and Qiangbin Wang†* †

Suzhou Key Laboratory of Nanomedical Characterization, Division of Nanobiomedicine and i-

Lab, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123 (China) ‡

College of Biological Science and Technology, Fuzhou University, Fuzhou, 350108 (China)

KEYWORDS: Tobacco Mosaic Virus, self-assembly, disulfide bond, nanorod

ABSTRACT: Tobacco mosaic virus (TMV) is a classical viral nanoarchitecture that has been extensively employed as a promising template for the fabrication of novel nanomaterials and nanostructures. Despite being an ideal source, the E. coli-derived TMV nanorod is suffering from tenuous assembly capability and stability. Inspired by the disulfide bond widely employed in biosystems, here we rationally introduce a cysteine into TMV coat protein (TMV-CP) to enable disulfide bond formation between adjacent subunits, thereby radically altering the behaviors of original noncovalent assembling system of wild type TMV-CP. The dramatically enhanced selfassembly capability and stability of the engineered TMV nanorods are observed and the essential

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roles of disulfide bonds are verified, illustrating a promising strategy to obtain desired geneticmodified nanorods which are inaccessible in plants. We expect this work will benefit the development of TMV-based nanotechnology and encourage the utilization of disulfide bond in other biomacromolecules for improved properties as nanoscaffolds.

INTRODUCTION

Biologically derived materials have attracted substantial attention in the fabrication of nanomaterials due to their distinct advantages such as well-defined structure, controllable assembly, genetically directed synthesis, easy manipulation and functionalization by biochemical protocols.1-5 Specifically, viruses and viral nanoparticles (NPs) have been effective candidates for components or templates to construct novel functional nanostructures and nanodevices within the realm of bioimaging,6, 7 biomedicine,8 nanoelectronics,9 energy,10,11 and catalysis.12 Among the established viral based nanoplatforms, tobacco mosaic virus (TMV) has been widely exploited as a template to construct desirable nanoarchitectures in recent years.13-17 TMV is a plant virus composed of a helical RNA and 2130 identical coat proteins (TMV-CP). Native TMV has a tube-shaped particle with 300 nm in length, an outer diameter of 18 nm and an inner channel of 4 nm in diameter. The directed self-assembly of TMV-CP into an ordered helical rod has brought a wealth of applications in biomineralization,18-21 patterned template assembly,22,23 cell culture,24,25 digital memory device,26 microbattery electrodes,27,28 artificial nanoenzymes,29 etc. We note that most of TMV rods in those works are obtained from tobacco plants. TMV virion derived from plants can retain structural integrity in a wide range of buffer conditions (pH 3.5-9), due to the binding of encapsulated genomic RNA to the coat proteins which strongly stabilizes the overall virus structure.30-33 However, the production of TMV particles in plants is


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limited by field conditions, growing season/growth-room conditions,34 complicated cultivation and time consuming.34,35 More importantly, genetically modified TMV mutants for predesigned functionality may suffer from failure of propagation in plants.36,37 To circumvent these drawbacks, heterologous expression systems have been tested for the production of TMV-CP for many years.37-39 Although with a high yield expressed in Escherichia coli (E. coli), the assembly of wild type TMV coat protein (WT-TMVP-CP) in the absence of RNA is sensitive to solvent pH, ionic strength and temperature as presented in Figure 1A, which considerably limits the wide applications of TMV. For instance, WT-TMVP-CP forms virus-like rods around pH 5.5 and nanodisks around pH 7.0 at room temperature (RT), while the nanorods/nanodisks will disassemble into A-proteins (an equilibrium between monomers and oligomers) at pH 8.0 (Figure 1A) or at 4 oC.33,40-42 Although efforts have been devoted to improve the in vitro assembly of TMV-CP by genetic engineering17,43 or chemical modification,44 there is no report of a simple and efficient method leading to recombinant TMV-CP assembly into stable nanorod, with minimal alterations to the protein.


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Figure 1. (A) Diagram of the preferred structures of WT-TMV-CP in the absence of RNA under different pH and ionic strength conditions at RT, which usually form nanorods in pH ranging from 5 to 7. Adapted with permission from Macmillan Publishers Ltd: Nature New Biology42, copyright 1971. (B) Scheme of nanorod assembled from T103C-TMV-CP in this study. Sitedirected mutagenesis of T103 to Cysteine is performed to achieve the recombinant TMV-CP. The assembly of T103C-TMV-CP is enhanced by disulfide bond formation in the low-radius region, and the resultant nanorods are stable over a much wider pH range from 5 to 11. Disulfide bond has been widely employed as the covalent linkage in nature to form stable protein nanostructures and switch protein functions.45,46 Herein, we report that the formation of disulfide bond between the adjacent subunits in the nanorods by introducing a cysteine by a single mutation of TMV-CP significantly improves the assembly capability and the stability of


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the nanorods formed from TMV-CP which is readily obtained from E. coli (Figure 1B). According to the structure of TMV-CP, the threonine at position 103 (T103) in the low-radius region of TMV is replaced by cysteine, which enables the formation of disulfide bonding between the adjacent cysteines and further stabilizes the nanorod structure. The in vitro assembly of the mutant protein is characterized under various conditions in comparison with WT-TMVCP, and the greatly enhanced assembly capability of the mutant protein has been observed. Furthermore, the structural stability of the resultant nanorods was examined in a wide pH range since the stability is a vital factor when protein nanostructures are used as nanoscaffolds. All the results illustrate the important role of disulfide bond in the assembly and stabilization of the TMV nanorods. The robustness of E. coli-derived new TMV-CP in assembly and stability would enable a versatile viral nanoplatform and impact the design and fabrication of TMV-based nanomaterials.

EXPERIMENTAL SECTION

Construction of TMV-CP expression plasmids. A wild type TMV-CP (WT-TMV-CP) gene optimized for the codon usage of E. coli was purchased from Sangon Biotech, Shanghai. The mutagenesis of T103C-TMV-CP was made by overlap PCR using two pairs of primers (P1: 5'AGATATACATATGAGCTATAGCAT-3' and P2: 5'-GTTTCCGCGGTACACGGATTGGC CT-3'; P3: 5'-AGGCCAATCCGTGTACCGCGGAAAC-3' and P4: 5'-GGAATTCTCAGGTCG CCGGGCC-3'). The gene of T103C-TMV-CP was then cloned into the pET32a(+) vector (Novagen) at NdeI-EcoRI restriction sites using T4 DNA ligase. The construct was verified by sequencing (Shanghai Invitrogen Biotechnology Co., Ltd.) and transformed into E. coli BL21 (DE3) strain (Novagen).


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Expression and purification of recombinant TMV-CP. As a general procedure, E. coli BL21 (DE3) containing the desired expression plasmid was cultured in Luria-Bertani medium containing 100 µg/mL ampicillin at 37 °C with constant shaking at 200 rpm. When cultures reached an optical density of 0.5 to 0.7 determined by OD at 600 nm, isopropyl β-D-1thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM. Then the cultures were grown for 8—12 h at 30 °C, harvested by centrifugation, and stored at -80 °C for further purification. Cells from a 500 mL culture were thawed, resuspended in 40 mL lysis buffer A (20 mM TrisHCl, pH 7.2, 20 mM NaCl, 20 mM EDTA) containing 15 mM dithiothreitol (DTT) and 1% protease inhibitor cocktail (Sangon Biotech, Shanghai), and then lysed by sonication. The resulting lysate was cleared by centrifugation for 30 min at 12,000 rpm. The cleared lysate was first purified by ammonium sulfate fractionation. Then the TMV-CP precipitate was resuspended in Phosphate Buffer (PB) (50 mM, pH 9.5) containing 20 mM DTT, followed by dialyzing against Bis-Tris buffer (20 mM, pH 6.6). The resulting protein solution was applied to a strong anion exchange column (HiTraP Q HP, GE Healthcare) on an AKTA prime plus FPLC system (GE Healthcare) and eluted with a 0—800 mM NaCl gradient. The elution buffer containing target protein was dialyzed against PB (50 mM, pH 9.5) and added with 20 mM DTT to prevent thiol oxidation. Purity was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified TMV-CP was flash-frozen and stored at -80 °C. Assembly of TMV-CP. For in vitro self-assembly in different conditions, TMV-CP protein solutions (protein concentration: 1.2 mg/mL) were dialyzed overnight at 20 °C against sodium acetate buffer (100 mM, pH 5.0), PB (400 mM, pH 7.0), PB (200 mM, pH 7.0), and PB (200 mM, pH 8.0), respectively, followed by several days of incubation for assembly. The assembly


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products of TMV-CP samples were examined by transmission electron microscopy (TEM) and agarose gel electrophoresis. More than 150 particles of each sample were measured for statistical analysis of nanorod length. Separation of T103C-TMV-CP nanorods and nanodisks by sucrose density gradient centrifugation (SDGC). Assembly products of T103C-TMV-CP in PB (400 mM, pH 7.0) containing nanorods and nanodisks were loaded onto a 10%—50% sucrose density gradient, and then centrifuged at 38000 rpm (SW40 Ti rotor, Beckman) at 15 °C for 3.5 h. Gradients were collected in 9 fractions from top to bottom by the marks on the SDGC tube. Aliquots were dialyzed against PB (100 mM, pH 7.0) and subjected to TEM examination. Stability analysis of T103C-TMV-CP nanorods at different pH values. After purified by SDGC, T103C-TMV-CP rods (0.7 mg/mL) were dialyzed against a series of 100 mM buffer solutions with various pH values of 7.0 (Na2HPO4-NaH2PO4), 9.5 (Na2CO3-NaHCO3), 10.0 (Na2CO3-NaHCO3), 10.5 (Na2CO3-NaHCO3), 11.0 (Na2HPO4-NaOH) and 11.5 (Na2HPO4NaOH). After 4 days of equilibration at 20 °C, the samples were characterized by TEM and agarose gel electrophoresis. More than 200 particles of each sample were measured for statistical analysis of nanorod length. DTT treatment. To investigate the assembly associated with DTT, T103C-TMV-CP sample (protein concentration: 1.2 mg/mL) was dialyzed overnight against PB (400 mM, pH 7.0) containing 100 mM DTT and incubated for 5 days at 20 °C. As a control, the same measure was performed in PB (400 mM, pH 7.0) without DTT. As for the evaluation of the effect of DTT treatment on the stability of T103C-TMV-CP rods, firstly, the rods (0.41 mg/mL) obtained by SDGC were exchanged into PB (200 mM, pH 7.0). Next, DTT was added to a final concentration of 0 mM, 0.01mM, 0.05 mM, 0.1 mM, 0.5 mM, 1 mM, 10 mM and 100 mM, respectively. All samples were incubated at 20 °C for 24 h and then were analyzed and with TEM and agarose gel


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electrophoresis. More than 200 particles of each sample were measured for statistical analysis of nanorod length. Agarose gel electrophoresis. TMV-CP samples (10 µL) added with ficoll at a final concentration of 4% were loaded into 3% agarose gels in Tris-acetate-EDTA (TAE) buffer. The electrophoresis was run for 50 min at 100 V. The protein bands in the gel were visualized by staining with Coomassie Brilliant Blue R-250 (Sangon Biotech, Shanghai). Transmission electron microscopy. A 10 µL portion of protein solution was applied to a carbon-coated copper grid, removed after 5 min with filter paper, and negatively stained for 3 min with 2 % phosphotungstic acid. All Samples were imaged on an FEI Tecnai 20 TEM operated at 200 kV. Images were recorded with a Gatan Ultra Scan 894 CCD camera and processed with the ImageJ software.

RESULTS AND DISCUSSION

Since the disulfide bond can only be formed between the proximal thiol groups, the selection of the cysteine mutation site is crucial to ensure the formation of disulfide bond within available distance. Based on the structure of TMV-CP (PDB ID: 2OM3) (Figure 1B), the T103 is considered as the target site since it locates at a junction of an α-helix and a loop region, which is near enough (ca. 0.8 nm) for two neighboring subunits to form a disulfide bond. In addition, the mutation site of T103 helps refrain from potential distortion of the TMV nanostructure and avoid the disordered inner segment which may handicap the contact of adjacent cysteines (Figure 1B). Furthermore, threonine is analogous to cysteine, which confirms the minimal effect of the mutagenesis on the electrostatic and hydrophobic interactions within TMV. In order to maximize the protein yield, a WT-TMV gene optimized for the codon usage of E. coli was used.47 The site-


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directed mutagenesis of T103C (Threonine 103 to Cysteine) gene is performed by standard recombinant techniques, and the target protein T103C-TMV-CP is successfully expressed and purified (Figure S1). To testify the assembly performance of T103C-TMV-CP, the self-assembly experiment was executed under the condition (100 mM sodium acetate buffer, pH 5.0, see Figure 1A) known to produce nanorods for WT-TMV-CP. In brief, WT-TMV-CP and T103C-TMV-CP were dialyzed against sodium acetate buffer (100 mM, pH 5.0) overnight at 20 °C, followed by two additional days of incubation. TEM observation (Figure S2) showed that both WT-TMV-CP and T103CTMV-CP could assemble into nanorods, accompanied with a number of nanodisks as expected.39 Interestingly, the nanorods assembled from T103C-TMV-CP were longer in length and higher in yield than those from WT-TMV-CP (Figure S2), implying the enhanced assembly capability of T103C-TMV-CP into nanorods. As illustrated in Figure 1A, the WT-TMV-CP phase diagram depicts that the higher pH and lower ionic strength of the buffer solution are unfavorable for its self-assembly into nanorods. For example, no nanorod is formed when the pH of the buffer solution is higher than 7.0 and the ionic strength is lower than 400 mM. To further systematically evaluate the self-assembly behaviors of T103C-TMV-CP, a series of experiments have been performed in harsh conditions in which WT-TMV-CP can not assemble into nanorods. The purified WT-TMV-CP and T103C-TMV-CP samples were dialyzed against 400 mM PB pH 7.0 overnight at 20 °C, followed by different incubation times. After collecting the products, an exciting phenomenon was observed that nanorods were assembled from T103C-TMV-CP at the third incubation day (Figure 2E) and more elongated nanorods were obtained with incubation going on (Figure 2F), while no nanorod formed from WT-TMV-CP even with the incubation time up to 5 days (Figure 2B,C). To further analyze the self-assembly of TMV-CP, agarose gel


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electrophoresis was performed. The products from different assembly stages can be visualized by staining with Coomassie Brilliant Blue R-250 after separation by electrophoresis. As shown in Figure 2A, the agarose gel visualized the assembly behaviors of WT-TMV-CP and T103C-TMVCP. In detail, the products from the dialyzed WT-TMV-CP and T103C-TMVP-CP samples without incubation resulted in two similar bands (Figure 2A, lanes 1 and 3), which corresponded to the nanodisks as examined with TEM (Figure 2B,D). For WT-TMV-CP, the product collected at the fifth incubation day (lane 2) ran as quick as the band in lane 1, indicative of the nanodisk structures as verified by TEM images in Figure 2C, which is consistent to the phase diagram in Figure 1A. In contrast, the dialysis product of the T103C-TMV-CP sample at the third incubation day presented a slower smear band (Figure 2A, lane 4), implying the occurrence of the assembly of T103C-TMV-CP into larger structures. TEM image of the sample of lane 4 (Figure 2E) shows that T103C-TMV-CP was assembled into nanorods with most of the length from tens nanometer to 250 nm (Figure S3A). Notably, the length of the nanorods assembled from the mutant protein was gradually elongated as incubation time goes on, as illustrated by the much slower migration band of the T103C-TMV-CP collected at the fifth incubation day (Figure 2A, lane 5) and the distribution of the elongated nanorod length from tens nanometer to 400 nm, with the maximum length up to 700 nm (Figure 2F and Figure S3B). These results illustrate that the self-assembly efficiency of T103C-TMV-CP is improved by introducing the disulfide bond. Furthermore, when executed in a lower ionic strength of PB (200 mM, pH 7.0) T103C-TMV-CP possessed the effective self-assembling capability with a longer incubation time in accord with the weakened electrostatic screening effect (Figure S4). In addition, previous report41 has demonstrated that WT-TMV-CP exists as the monomer state (A-protein) at pH 8.0, let alone nanodisks and nanorods. However, T103C-TMV-CP could not only assemble into nanodisks rapidly, but also assemble into nanorods after 20 days of incubation (Figure S5).


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Figure 2. The self-assembly of T103C-TMV-CP and WT-TMV-CP incubated in PB (400 mM, pH 7.0) for various times at 20 °C. (A) 3% agarose gel of the different products assembled from T103C-TMV-CP and WT-TMV-CP. WT-TMV-CP samples were shown in lanes 1 and 2, which were obtained after 0 day and 5 days of incubation, respectively. T103C-TMV-CP samples were shown in lanes 3, 4 and 5 which were incubated for 0 day, 3 days and 5 days, respectively. (B-C) TEM images of WT-TMV-CP samples incubated for 0 day and 5 days, respectively. (D-F) TEM images of T103C-TMV-CP samples incubated for 0 day, 3 days and 5 days, respectively. Sturtevant et al. has reported that the self-assembly of TMV-CP is endothermic and is affected by hydrophobic interactions.48 When placed at low temperatures, WT-TMV-CP nanorods and nanodisks are rapidly disassembled into A-proteins.41, 48 Does the mutated cysteine in T103CTMV-CP help to overcome the disassembly of the nanodisks and nanorods at low temperatures? The assembly behavior of T103C-TMV-CP at 4 °C in PB (400 mM, pH 7.0) was explored. After dialysis against PB (400 mM, pH 7.0) overnight, nanodisks were formed in both the WT-TMV-


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CP and T103C-TMV-CP samples (Figure 2B,D), and then the two samples were stored at 4 °C for a certain time. As expected, the WT-TMV-CP nanodisks were disassembled into oligomers in 3 days (Figure 3A). In contrast, T103C-TMV-CP sample not only resisted the disassembly of the nanodisks into oligomers originating from the low temperature, but also remained the selfassembly property which led to the formation of nanorods similar to those incubated at 20 °C after either 3 or 5 days (Figure 2E,F). Lasting the incubation time to one month, nanorods of T103C-TMV-CP with length more than 1 µm were obtained (Figure 3C,D), whereas no rod structure was observed in WT-TMV-CP sample (Figure 3B). Taken together, these observations convince us that the introduced cysteine dramatically prompts the self-assembly of TMV nanodisks into nanorods and stabilizes the as-formed nanorods.

Figure 3. The fate of nanodisks of WT-TMV-CP and T103C-TMV-CP at 4 oC. (A) The nanodisks of WT-TMV-CP were disassembled into irregular forms after 3 days. (B) TEM result of WTTMV-CP incubated at 4 oC for 30 days with no nanorod structure observed. (C) The nanodisks of T103C-TMV-CP were assembled into nanorods after 30 days incubation at 4 oC. (D) Histogram of the length distribution of the resultant nanorods in (C). In brief, we have demonstrated that the rationally introduced mutation of WT-TMV-CP into T103C-TMV-CP significantly alters the behaviors of original noncovalent assembling system of WT-TMV-CP and greatly improves the self-assembly of nanodisks into nanorods. However, as a classical template for nanomaterial fabrication, TMV nanorods may confront with various


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complicated surroundings. Structural stability is one of the critical parameters that determine the fate of the TMV nanorods in practical applications. Therefore, the stability of the T103C-TMVCP nanorods was systematically assessed in this work. The stability of the obtained T103C-TMV-CP nanorods was examined over a wide range of pH, which was carried out by dialyzing the nanorods against various buffer solutions after they were purified from the assembly mixture through SDGC (Figure S6). After equilibration with different pH buffer solutions for 4 days, all samples were characterized under TEM and agarose gel electrophoresis. It was found that the T103C-TMV-CP nanorods stayed intact in buffers with pH increasing from 7.0 to 11.0 (Figure 4), which is well known as unfavorable conditions for the WT-TMV-CP nanorods (Figure 1A). The length distributions of the nanorods under TEM images show no significant difference among the samples with various pH values, illustrating the high structural stability of T103C-TMV-CP nanorods highlighted by the greater resistance to alkaline induced disassembly. With the increase of pH value to 11.5, a faint band running faster than the nanodisk control in the agarose gel electrophoresis turned up (Figure S7), indicating the occurrence of dissociation of nanorods under pH of 11.5. These results demonstrate the robust nanorod structures of T103C-TMV-CP, which survive the harsh conditions that the WT-TMVCP nanorods are disassembled and promises its wide applications in future nanostructure construction.


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Figure 4. The stability assessment of T103C-TMV-CP nanorods against different pH values. (A) Histogram of the length distributions of nanorods collected from the buffer solutions with various pH values. (B-F) TEM images of the samples in buffers with pH 7.0, pH 9.5, pH 10.0, pH 10.5, and pH 11.0, respectively. In order to verify the assumption that the significantly enhanced assembly capability and stability of T103C-TMV-CP are attributed to the formation of disulfide bonds between the introduced cysteines, non-reducing SDS-PAGE analysis was performed. After T103C-TMV-CP and WT-TMV-CP were incubated in PB (400 mM, pH 7.0) for three days, the samples were processed in loading buffer with or without DTT and were analyzed through SDS-PAGE. Samples treated with loading buffer containing DTT will generate only one band corresponding to the monomer, no matter whether they contain disulfide bonds or not, while samples treated with loading buffer without DTT can generate bands of dimer if there are disulfide bonds. As shown in Figure 5, after the electrophoresis, both T103C-TMV-CP and WT-TMV-CP in loading


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buffer containing DTT resulted in identical monomer bands referring to molecular weight of TMV-CP (17.5 kDa). However, the samples in non-reducing agent loading buffer behaved differently between WT-TMV-CP and T103C-TMV-CP. WT-TMV-CP generated one band at the position of 17.5 kDa, whereas T103C-TMV-CP resulted in a major band at the position around 35.0 kDa, matching the expected molecular weight of TMV-CP dimer. The results demonstrated that disulfide bonds are indeed formed between adjacent subunits of T103C-TMVCP in nanorods as we expected. It is interesting to note that two faint bands appeared in the sample of T103C-TMV-CP processed with non-reducing loading buffer. Besides that some TMV CP may not form any disulfide with other CP and will appear as a faint band in the gel, we expect it is likely due to asymmetric cleavage of the T103C-TMV-CP dimer when it was subjected to a fierce heating procedure of denaturation (in this case, 100 oC for 5 min) before SDS-PAGE (Figure S8).

Figure 5. SDS-PAGE of TMV-CP to demonstrate the dimer of T103C-TMV-CP containing disulfide bond. Lane 1, T103C-TMV-CP sample mixed with loading buffer containing DTT. Lane 2, T103C-TMV-CP sample mixed with loading buffer without DTT. Lane 3, WT-TMV-CP


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sample mixed with loading buffer containing DTT. Lane 4, WT-TMV-CP sample mixed with loading buffer without DTT. The numbers on the left indicate the molecular weights in kDa representing the protein standard markers. Non-reducing SDS-PAGE results (Figure S9) may provide extra clue to explain the formation of disulfide bond during the self-assembly of T103C-TMV-CP in PB (400 mM, pH 7.0) (Figure 2D-F). After dialysis overnight (0 day), the disk structure has been formed from T103C-TMV-CP (Figure 2D), which is similar to WT-TMV-CP assembly (Figure 2B). After the assembly of disk, the distance between two mutated cysteines is close enough for the formation of disulfide bond. Then the disulfide bonds were quickly formed during the assembly process of rod from 0 day to 3 days (Figure S9). Futhermore, little alteration has been observed with elongating incubation time to 5 days, indicating most of disulfide bond formed after 3 days of incubation. This self-assembly behaviour is quite different from WT-TMV-CP (Figure 2). WT-TMV-CP in vitro is highly polymorphic and contingent on the different solvent conditions like pH and ionic strength (Figure 1A), due to its innate physicochemical property of protein that involves hydrophobic interactions, electrostatic interactions, hydrogen bonds, etc.33,41,42 The nanodisks assemble from TMV-CP subunits much more readily than nanorods and further aggregate into the rods when changing to a favorable condition.40,42 This spontaneous process is reversible upon variation of buffer conditions. Increasing ionic strength and lowering pH value of buffer have a positive effect on the assembly of rods. The former causes an electrostatic screening effect, while the later reduces the net negative charge of proteins, both leading to the lowering of electrostatic repulsion between disks thereby being in favor of the assembly. In addition, the assembly of TMV-CP is endothermic and is driven by hydrophobic interactions.41,48 WT-TMV-CP at low temperature will be in disassembly state as a result of entropically driven


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effect. In the case of our mutated T103C-TMV-CP, it possesses quite similar physicochemical properties to TMV-CP due to the only single mutation from threonine to cysteine, resulting in a similar assembly process to WT-TMV-CP except the formation of disulfide bond. The irreversible formation of disulfide bond stabilizes the binding between subunits and breaks the equilibrium of original system (WT-TMV-CP) to favor the rod formation. Therefore, on the one hand, increasing ionic strength and lowering pH value benefit the assembly of T103C-TMV-CP into rods. On the other hand, in contrast to WT-TMV-CP, the formed disulfide bonds between adjacent disks can still prompt the T103C-TMV-CP to assemble into rods and retain high stability under harsh conditions such as pH 7.0, 400 mM PB at 4 oC. To further corroborate that disulfide bond directly contributes to the assembly of nanorods, the assembly of T103C-TMV-CP in the presence of DTT was carried out in PB (400 mM, pH 7.0) for 5 days of incubation. Agarose gel electrophoresis (Figure 6A) showed that the assembly of T103C-TMV-CP in the presence of DTT generated a band (Lane 3) close to the disk control (Lane 1), quite different from the assembly without DTT (Lane 2). TEM observation confirmed that the assembly in the presence of DTT dominantly produced disk structures (Figure 6C), while the sample incubated without DTT obtained nanorods of several hundred nanometer in length (Figure 6B). Therefore, the assembly of rods from T103C-TMV-CP can be significantly inhibited by the reducing agent DTT, confirming the essential role of disulfide bond in the TMV nanorod assembly process. Taken together, the formation of disulfide bond is thought to covalently link the adjacent subunits to form the nanorods and further stabilize the nanorods.


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Figure 6. Interruptive roles of DTT in the assembly of T103C-TMV-CP in PB (400 mM, pH 7.0). (A) Visualization of assembly products in agarose gel image. T103C-TMV-CP containing 100 mM DTT (lane 3) showed a band being close to disk control (lane 1) in comparison to the sample without DTT (lane 2). (B) TEM image of T103C-TMV-CP incubated in PB (400 mM, pH 7.0) for 5 days. (C) TEM image of T103C-TMV-CP incubated in PB (400 mM, pH 7.0) containing 100 mM of DTT for 5 days, where nanodisks were the dominant species in the sample. Finally, DTT treatment was executed to evaluate the contribution of disulfide bonds to the stabilization of the nanorods. T103C-TMV-CP nanorods purified through SDGC were treated with DTT of a series of concentrations from 0 to 100 mM for 24 h (protein concentration: 0.41 mg/mL). As shown in the agarose gel image (Figure 7A), the products treated with DTT moved faster than the original nanorods but slower than the control of nanodisks, indicating the nanorods are dissociated into shorter ones as the DTT concentration increases. In agreement, TEM analysis showed the shortening effect of DTT on the nanorods (Figure 7C-E). For example, the length of the T103C-TMV-CP nanorods without DTT treatment dominated in the range of 150 to 199 nm, while the length peaks of the nanorods treated with 0.1 mM DTT and 100 mM DTT shifted to 100-149 nm and 50-99 nm, respectively (Figure 7B). These results evidence the


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considerable contribution of the disulfide bond to the stabilization of the TMV nanorods. In addition, densitometry analysis of the gel electrophoresis results (Figure S10) showed that the amount of T103C-TMV-CP protein in the form of nanorods remained constant in all the samples treated with different concentrations of DTT, in comparison with the original nanorods, suggesting that the nanorods preferred to crack in the internal section, rather than be disassembled gradually from the ends during DTT treatment. On the other hand, these observations also showed that the T103C-TMV-CP nanorods remained their rod morphology (only with the shortening effect) and were not dissociated into nanodisks even at a high DTT concentration of 100 mM. It has been reported that the conformational change in the low-radius region of TMV-CP can function as an important regulatory mechanism in virus assembly/disassembly.49-51 The disordered low-radius inner loop region (residues 88-109) can promote the assembly of nanorods if they are stabilized by RNA binding.50, 51 The disulfide bond introduced in our T103C-TMV-CP is thought to have a similar effect on stabilizing the lowradius region. Therefore, the formation of T103C-TMV-CP nanorod may achieve an ordered conformational change in the low-radius region, leading to a higher stability upon the synergetic effect of disulfide bonds and other factors, such as electrostatic forces, hydrogen bonds, and hydrophobic patch.51 In the case of the DTT treatment, the synergistic effect of these interactions explains the resistance of T103C-TMV-CP nanorod against the disassembly triggered by DTT. Additionally, the length dependence of the nanorods on DTT concentration may afford us an effective avenue to tune the length of TMV nanorods.


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Figure 7. The stability assessment of T103C-TMV-CP nanorods against DTT treatment. (A) Agarose gel image of the T103C-TMV-CP nanorods against different concentration of DTT. The first lane in left shows the control of nanodisks. From left to right, the second lane to the last lane correspond to samples processed under different DTT concentrations. (B) Histogram of the length distributions of T103C-TMV-CP nanorods with DTT concentrations of 0 mM, 0.1 mM, and 100 mM, respectively. (C-E) TEM images of the products collected from the samples with DTT concentration of 0 mM, 0.1 mM, and 100 mM, respectively.

CONCLUSIONS

We have demonstrated that the in vitro assembly and stability of nanorod structures from TMV-CP generated in E. coli can be notably enhanced by rationally mutating T103 into cysteine, which can form disulfide bond between adjacent subunits. Gel electrophoresis and TEM


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observation consistently show that the mutant TMV-CP can efficiently self-assemble into nanorods in both acid and alkaline buffer solutions, and moreover, the engineered nanorods can keep stable in buffers over a much wider pH window from 5 to 11, whereas the wild type TMVCP can only form nanorods and remain its nanorod structures in acid buffers. Non-reducing SDSPAGE and DTT treatment assay confirm the essential roles of disulfide bonds in the selfassembly and stabilization of the engineered TMV nanorods. Such engineered TMV-CP represents a new version of TMV protein with outstanding self-assembly capability, robust rod structures, easy genetic-engineering, and massive preparation, which makes it an intriguing candidate for protein-nanorod-based material development. The present work can serve as a paradigm of controlling protein assembly behaviors by rational design with disulfide bond and thereby the enhanced structural properties.

ASSOCIATED CONTENT Supporting Information Supplementary results and discussion, SDS-PAGE analysis of the purification of TMV-CP, TEM images of the assembly products in sodium acetate buffer and PB, separation of T103CTMV-CP nanorods and nanodisks by SDGC, agarose gel electrophoresis analysis of the stability assessment, non-reducing SDS-PAGE result of the assembled T103C-TMV-CP samples heated with different time lengths from 1 min to 20 min before electrophoresis, non-reducing SDSPAGE result of the T103C-TMV-CP incubated in PB (400 mM, pH 7.0) from 0 day to 5 days, and densitometry analysis of the gel electrophoresis results. This material is available free of charge via the Internet at http://pubs.acs.org.


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AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

This work is supported by the CAS “Bairen Ji Hua” program, NSFC (31271076, 91023038), Jiangsu Province NSF (BK2012007), MOST (2011CB965004), and CAS/SAFEA International Partnership Program for Creative Research Teams. The authors thank Prof. Q. Wang at University of South Carolina for providing the TMV plasmid. REFERENCES 1.

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