F t R a b c a A R R A A K V V E P 1 o [ 5 t g s v t f t b e V a i c n t s h 0 International Journal of Biological Macromolecules 83 (2016) 178–184 Contents lists available at ScienceDirect International Journal of Biological Macromolecules j ourna l ho me pa g e: www.elsev ier .com/ locate / i jb iomac unctional expression, monodispersity and conformational changes in he SBMV virus viral VPg on binding TFE .B. Mariutti a, I.P. Carusob, A. Ullaha, F.R. De Moraisb, D. Rehdersc, R.K. Arnia,b,∗ Multiuser Center for Biomolecular Innovation, IBILCE/UNESP, Brazil Department of Physics, IBILCE/UNESP, Brazil Laboratory for Structural Biology of Infection and Inflammation, Hamburg University, Germany r t i c l e i n f o rticle history: eceived 14 July 2015 eceived in revised form 8 November 2015 ccepted 10 November 2015 vailable online 22 November 2015 a b s t r a c t Southern bean mosaic virus (SBMV) RNA purified from infected plants was used for cloning the viral genome-linked protein (VPg) and was subsequently expressed in Escherichia coli. Circular dichroism (CD), dynamic light scattering (DLS) and saturation transfer difference (STD) by nuclear magnetic resonance (NMR) measurements were employed to determine the degree of monodispersity and to investigate the eywords: Pg iral genome-linked protein xpression urification conformational changes in the absence and presence of trifluoroethanol (TFE) which indicated increased helical content with increasing concentration of TFE. 8-Anilino-1-naphthalenesulfonic acid (ANS) was used as a probe to compare the unfolding regions of the protein before and after addition of TFE. The results indicated that although the TFE concentration influences VPg folding, it does not play a role in nucleotide binding and that the local solvent hydrophobicity causes significant conformational changes. © 2015 Elsevier B.V. All rights reserved. . Introduction Genome-linked viral proteins (VPgs) are involved in a number f processes ranging from replication to viral protein synthesis 1,2]. VPgs are small proteins that are covalently linked to the ′ end of viral RNA via a phosphodiester bond formed between he hydroxyl groups of amino acid residues and the 5′ phosphate roups of RNA [3,4]. Often encountered in viruses with single- tranded positive-sense RNA (ssRNA) genomes, VPgs from fungal iruses, plant viruses and animal viruses with double or posi- ive single strand (ssRNA) have been characterized [5,6]. VPgs rom plant and animal viruses share many features, for example, hey are products of polyprotein processing and are uridylated y their cognate RNA-dependent RNA polymerase (RdRP) [7–9] nabling VPgs to operate as primers during viral RNA synthesis. Pgs also share the presence of high percentages of basic amino cids (mostly lysine, glycine, threonine and arginine) contribut- ng to the interaction with the negatively charged RNA [10]. The ovalent binding of VPgs to RNAs exhibits some differences; picor- aviruses, potyviruses and caliciviruses use the hydroxyl group of a yrosine residue whereas comoviruses and nepovirus are reported ∗ Corresponding author at: Multiuser Center for Biomolecular Innovation, Univer- idade Estadual Paulista (UNESP), São Jose do Rio Preto, 15054-000 SP, Brazil. E-mail address: arni@sjrp.unesp.br (R.K. Arni). ttp://dx.doi.org/10.1016/j.ijbiomac.2015.11.026 141-8130/© 2015 Elsevier B.V. All rights reserved. to use a serine residue [6]. Threonine also contains a hydroxyl group, but the only evidence that it is used for RNA binding was reported by [11] when investigating VPg from SBMV covalently attached to genomic RNAs. VPg from sobemovirus is a cleavage product of its precursor polyprotein (VPg-proteinase-polymerase) [12] and the residue for RNA binding is not conserved within its genre [13]. All positive-sense ssRNA viruses that infect mam- malian, insect or plant cells replicate in association with host endomembrane [14] and different host factors may influence the process [15,16]. Some phytoviral (Sesbania mosaic virus; Potato virus A, Potato virus Y, Lettuce mosaic virus, and Rice yellow mottle virus) VPgs were shown to be natively unfolded proteins [17–20] and hence recalcitrant for crystallization. Therefore, to date, not a single crystal structure of these proteins has been determined. Nevertheless, the crystal structures of synthetic peptides corre- sponding to Picornaviridae VPgs (<3 kDa) (PDB: 2D7S, 4IKA, 3CDW) complexed with their cognate RdRP indicate that the peptides are almost completely random coiled without any alpha helix or beta strand. More recently, the solution structure of recom- binant VPg from Caliciviridae (PDB: 2MXD) has been elucidated [21]. In the present study, VPg from SBMV was expressed in Escherichia coli and purified. Circular dichroism (CD) was employed to assess the VPg secondary structure conformational content and its variation in the presence of increasing con- centrations of reagents that mimic membrane environments dx.doi.org/10.1016/j.ijbiomac.2015.11.026 http://www.sciencedirect.com/science/journal/01418130 http://www.elsevier.com/locate/ijbiomac http://crossmark.crossref.org/dialog/?doi=10.1016/j.ijbiomac.2015.11.026&domain=pdf mailto:arni@sjrp.unesp.br dx.doi.org/10.1016/j.ijbiomac.2015.11.026 f Biolo ( n r s N b u 2 2 g t i 2 O t a r s t t A a ( T p i o T a T i a t a 1 o P ( v 2 v E c c O a 2 B g a R.B. Mariutti et al. / International Journal o 2,2,2-trifluoroetahnol, TFE). Far UV-CD spectra and 8-anilino-1- aphthalenesulfonic acid (ANS) were used to compare the unfolded egions of the protein before and after the addition of TFE and aturation transfer difference by nuclear magnetic resonance (STD- MR) spectroscopy was employed to compare the binding pattern etween the VPg from SBMV and dNTPs, to gain insights into molec- lar recognition of different nucleotides by VPg. . Methods .1. Virus purification and RNA extraction The virus was purified from infected leaves of Phaseolus vul- aris following the method of [22]. Viral RNA was extracted using he RNeasy Plant Mini Kit (Qiagen) following the manufacturer’s nstructions. .2. Primer design, cDNA syntheses and PCR amplification of the RF1 Primers were designed as shown below to amplify the sequence hat encodes VPg from genomic RNA of an isolate of SBMV. BamHI nd XhoI restriction sites were incorporated in the forward and everse primers, respectively, to facilitate cloning in pET28a expres- ion vector. A TEV protease cleavage site was also incorporated in he forward primer to permit cleavage of the hexahistidine tag of he recombinant protein after purification. Forward primer: 5′-GGATCCGGTGAAAATTTATATTTTCAAGGT- CTCTACCT CCTGATCTGTCCG-3′ (BamHI restriction site underlined nd TEV cleavage site in bold). Reverse primer: 5′-CTCGAGTCATTCCTGAGCTGAAGTCCA-3′ XhoI restriction site underlined). First strand cDNA was synthesized using Superscript II Reverse ranscriptase (Invitrogen). Polymerase chain reaction (PCR) was erformed for the amplification of the sequence that encodes VPg n 100 �L mixture containing 50 ng of genomic RNA, 1 �L (10 �M) f each primer, 2 �L (10 mM) dNTPs, 10 �L of PCR buffer (100 mM ris–HCl, pH 8.8, 500 mM KCl, 0.8% Nonidet P40, and 25 mM MgCl2), nd 2.5 U of native Taq DNA polymerase enzyme (MBI Fermentas). he reaction was carried out using the following reaction cycles n a programmable thermocycler (Eppendorf): initial denaturation t 95 ◦C for 10 min followed by 30 consecutive cycles of dena- uration at 95 ◦C for 30 s, annealing for 1 min at 55 ◦C, extension t 72 ◦C for 1 min 30 s, followed by a final extension at 72 ◦C for 0 min. The amplification product was analyzed by electrophoresis n a 1% agarose gel stained with ethidium bromide. The specific CR product obtained was purified using PCR gel purification kit Qiagen) and the product was used for ligation in pGEM-T Easy ector. .3. Cloning in pGEM-T easy vector In order to clone the sequence that encodes VPg, pGEM-T Easy ector (Promega) was used. The resulting PCR product and pGEM-T asy vector were ligated overnight at 4 ◦C using T4 DNA ligase. This onstruct was transformed into E. coli DH5� cells and the resulting olonies were screened by blue white colony selection and PCR. ne clone was used to plasmid extraction and it was sequenced by utomatic sequencer ABI 377 DNA Sequencer. .4. Cloning in pET28a The sequence cloned in pGEM-T Easy vector was digested with amHI and XhoI restriction enzymes and analyzed on a 1% agarose el. A 300 bp insert (ORF) was purified from the agarose gel by using gel extraction and purification kit (Qiagen) and cloned in pET28a gical Macromolecules 83 (2016) 178–184 179 vector (Novagen). Positive clones were first selected by PCR and reconfirmed by restriction digestion. 2.5. Expression of recombinant VPg in E. coli using pET28a vector For expression, VPg protein was tagged with 6xHis; E. coli cells BL21-CodonPlus®-RIL were transformed with the pET28a con- struct and incubated in LB broth (with 0.2% glucose and 34 �g/ml kanamycin for selection). The medium was inoculated with an overnight culture (1:100 dilution) and the culture was incubated under agitation at 30 ◦C until an OD600 of ∼0.5 was attained. Sub- sequently, 0.2 mM IPTG was added and the culture was further incubated at 18 ◦C for 16 h. 2.6. Protein purification 2.6.1. Purification of the VPg 6xHis recombinant protein Cells were harvested by centrifugation at 6000 × g, at 4 ◦C for 20 min and lysed in buffer 20 mM sodium phosphate pH 7.4, 200 mM NaCl (buffer A) by sonication on ice. The clear supernatant obtained by centrifugation at 15,000 × g for 45 min (4 ◦C) was sub- sequently applied onto a nickel resin column pre-equilibrated with buffer A. The column was washed with buffer A which addition- ally contained 70 mM imidazole and the recombinant protein was eluted with buffer A which contained 400 mM imidazole, con- centrated and subjected to a final step of molecular exclusion chromatography by utilizing a Superdex G75/300 GE column. The purity of VPg throughout E. coli expression and purification steps was determined by SDS-PAGE. 2.7. Dynamic light scattering DLS measurements were carried out using freshly prepared samples in buffer 20 mM sodium phosphate pH 7.5, 100 mM NaCl. Each experiment was carried out in a quartz cuvette with an optical path length of 3 mm at 25 ◦C and the results presented are the aver- age values obtained from 20 scans. The experiments were repeated at different pHs in the presence of salts, amino acids, sugars, deter- gents, reducing agents and also in the presence of possible ligands, such as, dATP, dUTP, dGTP and dCTP (Table 1). 2.8. Circular dichroism spectroscopy Far UV-CD spectra were recorded at room temperature (25 ◦C) on a Jasco J-710 Spectropolarimeter (Jasco, Tokyo, Japan) and quartz cells with a path length of 0.5 mm. CD spectra were recorded in the 190–260 nm range at a scan rate 50 nm/min, response time of 1.0 s, spectral bandwidth of 1.0 nm and spectral resolution of 0.1 nm. For each spectrum, 7 accumulations were performed. The VPg concen- tration was maintained constant at 30 �M during all experiments. The addition of 2,2,2-trifluoroethanol (TFE, Sigma) was performed in the 0–30% range with an increment of 10% (v/v). All spectra were corrected by subtraction of the respective buffer spectra. Secondary structure percentages for each tested condition were calculated with CONTINLL software of CDPro package, using the reference set of proteins SMP56 [23]. 2.9. Binding of probe 8-aniline-1-naphthalene sulfonate (ANS) The fluorescence measurements were performed at room temperature (25 ◦C) by using an ISS PC1 steady-state Spectrofluo- rimeter (Champaign, IL, USA) equipped with quartz cells of 10 mm path lengths. Both excitation and emission bandwidths were set at 8 nm. The excitation wavelength at 370 nm was chosen since it only causes excitation of ANS. The emission spectrum was collected in the range of 390–700 nm with an increment of 1 nm and each point 180 R.B. Mariutti et al. / International Journal of Biological Macromolecules 83 (2016) 178–184 Table 1 Reagents used to investigate the aggregation state of recombinant VPg. Reagents Concentration range Salts NaCl 0–1 M KCl 0–1 M NaF 0–500 mM NaCl + KCl 0–500 mM CaCl2 0–200 mM MgCl2 0–200 mM MgSO4 0–400 mM Urea 0–500 mM Amino acids Glycine 0.5–2% l-Arginine 0–0.5 M Genetic material Oligonucleotides 0.1–100 �M Nucleotides (dNTPs) 0–0.5 mM Sugars and alcohols �-Cyclodextrin 0–100 mM Sucrose 0–1 M Glucose 0–1 M Sorbitol 0–40% Glycerol 5–40% Trifluoroethanol 5–30% Ethanol 0–5% Detergents Triton X-100 0–0.02% Tween 20 0–0.1% CHAPS 0–0.5% NP-40 0–0.2% DTT 0–20 mM i A i s i 2 p d t 2 s H c a c s s p o + c t o r i A S t t Fig. 1. SDS-PAGE 12% gel of viral particles obtained from infected bean leaves: lane (M), molecular-weight markers (labelled in kDa); lane (1), represents purified SBMV. Fig. 2. SDS-PAGE analysis of purified VPg. Lane (M), molecular-weight markers (labelled in kDa). (A) tagged, purified VPg. (B) and (C) tagged VPg after 8 and 16 h of Reducing agents 2-Mercaptoethanol 0–10 mM n the emission spectrum is the average of 10 accumulations. The NS binding probe (250 �M) to VPg (100 �M) was monitored both n the absence and presence of 30% TFE (vol:vol). As a reference pectrum, the ANS emission was also analyzed in a buffer solution n the absence and presence of 30% TFE. .10. Prediction of intrinsic disorder in VPg VPg amino acid sequence was analyzed using computational redictor software FoldIndex© tool to identify ordered and disor- ered regions within the protein. The intrinsic disorder in VPg and he structural changes induced by TFE were evaluated. .11. Saturation transfer difference nuclear magnetic resonance pectroscopy NMR experiments were performed on a Brucker AVANCE III D 600 MHz NMR spectrometer equipped with a triple-resonance ryoprobe. The protein concentration was adjusted to 15 �M in 50 mM deuterated phosphate buffer pH 7.5 and the ligand oncentration was 300 �M. Saturation transfer difference NMR pectroscopy [24] experiments were carried out using the standard tddiffesgp.3 pulse sequence at 393 K, where protein signal sup- ression is performed by a spin-lock filter. For protein saturation, n-resonance radiation was set to +300 Hz and off-resonance to 24,000 Hz. Control experiments were performed under identical onditions in the absence of protein. To check whether conforma- ional changes can impair binding, experiments were also carried ut in the presence of 30% TFE under identical conditions. Group epitope mapping was performed by comparing off- esonance and difference spectra and adjusting the relative signal ntensities in the calculation of the STD amplification factor (STD- F) following the equation: TD-AF = Ioff-resonance − Ion-resonance Ioff-resonance The largest STD-AF is associated to 100% relative STD effect, and he others signals are compared to it. Higher STD-AF is associated o closer contact to protein atoms since the Nuclear Overhauser digestion using TEV protease. The black arrow indicates TEV protease, green arrow indicates untagged VPg and red arrow the tag. (D) Untagged VPg isolated. (E) 1 �L of VPg concentrated to 10 mg/mL. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) R.B. Mariutti et al. / International Journal of Biological Macromolecules 83 (2016) 178–184 181 F m) of t r E d 3 3 l t i s c D ig. 3. Dynamic light scattering analysis of VPg (A) a plot (20 scans) of the radius (n adius of VPg. The data are the average of 20 measurements. ffect (NOE) is proportional to the inverse of the sixth power of the istance between two nuclei. . Results and discussion .1. Virus purification, RNA extraction and cloning of VPg of SBMV High purity viral particles were obtained from infected bean eaves (Fig. 1), serving as a source of RNA for cDNA synthesis. In his study, the sequence that encodes VPg from SBMV was cloned nto the prokaryotic expression vector pET28a. Clones were first creened by PCR analysis and then the expression construct was hecked for in-frame fusion by restriction enzyme digestion and NA sequencing (data not shown). Fig. 4. Dynamic light scattering analysis of VPg indicating the estimated hydrodynam he particles in solution as a function of time (s), (B) estimated mean hydrodynamic 3.2. Purification and hexahistidine tag removal Recombinant VPg was purified using Ni-NTA affinity chro- matography and incubated with recombinant TEV protease for 16 h at room temperature. The hexahistidine tag and the TEV protease were removed using a second step of Ni-NTA affinity chromatog- raphy. The unbound VPg fraction was collected, concentrated to 10 mg/mL and its purity was verified by SDS-PAGE (Fig. 2). 3.3. Dynamic light scattering Since dynamic light scattering (DLS) provides information about the size distribution of particles in solution, concentrated VPg sam- ples in 20 mM buffer sodium phosphate pH 7.5 and 100 mM NaCl, ic radius of VPg is 5 nm in the presence of 1 mM dATP (A) and 30% of TFE (B). 182 R.B. Mariutti et al. / International Journal of Biological Macromolecules 83 (2016) 178–184 Fig. 5. Far UV-CD spectra of purified VPg in the absence (black line) or in the presence of 10% (red line), 20% (orange line) and 30% (yellow line) of TFE. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Table 2 Percentages of VPg secondary structure in the absence and presence of TFE. TFE (%) �-Helix (%) �-Sheet (%) Turn (%) Random coil (%) 0 8 31 24 37 10 8 33 23 36 20 9 35 23 33 w m h 1 o h c 3 p g a o b T t i i o t t 2 T o o a p t o o Fig. 6. Fluorescence emission spectra of the binding of ANS to VPg; in the absence (red line) and presence (yellow line) of 30% TFE, and the control ANS emission in STD-NMR spectroscopy was first described by Mayer and Meyer [24] and is widely used for ligand screening and affinity evaluation 30 15 32 22 31 ere present as a monodisperse population (Fig. 3A) with an esti- ated hydrodynamic radius of 10 nm (Fig. 3B). The presence of TFE or dATP in solution reduced the estimated ydrodynamic radius of the protein complex from approximately 0 to 5 nm (Figs. 4 and 5) contrary to that observed in the presence f the other additives tested. VPg is an elongated protein and the ydrodynamic radius of 50 Å indicates that it oligomeric under the onditions tested. .4. Circular dichroism Many intrinsically disordered VPgs such as those from oliovirus (genus Enterovirus), Rice yellow mottle virus (RYMV, enus Sobemovirus) and Lettuce mosaic virus, Potato virus A (LMV nd PVA, genus Potyvirus) undergo an increase in the percentage f �-helical content in the presence of natural and artificial mem- ranes and also in environments that mimic membranes such as FE [17]. The VPg CD spectrum in the absence of TFE displayed struc- ural characteristics of an intrinsically disordered protein since ts observed minimum was near 205 nm with a negative elliptic- ty at 190 nm (Fig. 5); the addition of TFE to the buffer solution f VPg induced folding into a predominantly �-helical conforma- ion. The increase in the �-helical content of VPg is based on he positive ellipticity at 190 nm, the shift of the minimum from 05 to 209 nm and increase of the negative ellipticity at 222 nm. able 2 presents the percentages of secondary structure of VPg btained using the CONTINLL software. The �-helix propensity f VPg changes with the increment of TFE from 8% to 15% as result of the stabilization of the �-helix and the random coil ercentage decreased from 37% to 31%. Other secondary struc- ural elements underwent only slight changes with the addition f TFE, suggesting that these structures constitute the stable core f VPg. phosphate buffer in the absence (black line) and presence (orange line) of 30% TFE in the absence of protein. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 3.5. Binding of probe ANS The binding of ANS probe to VPg was monitored by steady-state fluorescence spectroscopy. The dye ANS is a probe widely used to investigate the structural changes in proteins and this probe pos- sesses high affinity for the hydrophobic environments in proteins giving rise to significantly enhanced fluorescence with a charac- teristic blue shift of its emission maximum. Fig. 6 indicates that the fluorescence spectrum of ANS in phosphate buffer increased in the presence of VPg and there was a blue shift of the emis- sion maximum. This increase in the blue shift of the fluorescence emission is characteristic of the binding of ANS probe to unfolded proteins. On the other hand, the emission spectrum of ANS in the presence of VPg plus 30% TFE presented a minor change in rela- tion to its fluorescence in phosphate buffer plus 30% TFE, compared with results obtained in the absence of TFE. This minor change in fluorescence emission of ANS in the buffer with TFE is character- istic of folded proteins. The presence of TFE in the buffer solution likely induced the stabilization of the secondary structure of VPg, since TFE competes for hydrogen bonds with water molecules and thereby inducing the protein to form more hydrogen bond with itself. 3.6. Prediction of intrinsically unstructured/disordered sequence Using FoldIndex© tool, a predictor of intrinsically disordered regions in the protein sequences, we encountered a probable dis- ordered region of VPg (red sequence in Fig. 7), about 38% of the sequence is structured (green sequence in Fig. 7) in agreement with the circular dichroism experiments which indicated that 38% of the VPg was structured (8% �-helice + 31% de �-strand). Based on this analysis, we propose, that the conformational changes induced by TFE in VPg of SBMV occur principally in the central and C-terminal regions. 3.7. Nuclear magnetic resonance spectroscopy For the characterization of the binding interactions between VPg and deoxynucleotides triphosphates (dNTP), the technique of satu- ration transfer difference NMR (STD-NMR) spectroscopy was used. [25,26]. Ligand interactions are detected by selective radiofre- quency irradiation of only the protein signals in order to saturate R.B. Mariutti et al. / International Journal of Biolo Fig. 7. Bioinformatics based fold index values for VPg of SBMV. The fold index plot indicates the difference between the ordered (green) and disordered (red) regions based on the amino acid composition. The plot predicts extensive unfolded regions in the central and C-terminal regions of the protein. FoldIndex server http://bioportal. w fi i t e c s contribution of the C8-proton was significantly higher and from F S i eizmann.ac.il/fldbin/finde. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.) t, the so-called on-resonance spectrum. Magnetization is then ransferred to the ligand via the intermolecular nuclear Overhauser ffect and detected if the interaction takes place under the fast hemical exchange condition and interaction of the order of the dis- ociation constants from a few micromolar to tenths of millimolar ig. 8. STD-NMR spectroscopy of VPg and dNTPs. Difference spectra of (a) dATP, (b) dCTP, TD effects are evaluated by percentages compared to the higher individual signal of eac n dCTP, dGTP and dUTP while for dATP they are only 50%; indicating that the interaction gical Macromolecules 83 (2016) 178–184 183 may be detected. The key idea behind this technique is that small ligands have positive NOE signals while large macromolecules have negative NOEs. In addition, by comparing the signal intensities in the off-resonance and difference spectra it is possible to evaluate the group epitope mapping, i.e., the ligand atoms that are interact- ing closely in the protein binding site. Group epitope mapping is considered to be a great advantage of STD-NMR spectroscopy over other techniques used for studying protein–ligand interactions. This information may be used to highlight key differences in the ligand architecture that lead to stable protein interactions. In the present study, STD-NMR spectra indicated STD effects on the deoxyribose protons C1′, C2′ and C3′ as well as on the nucleobase protons (Fig. 8). Quantifying STD effects as relative STD percentages, the binding epitopes are further assigned. For all dNTPs a significant binding contribution of the deoxyribose moiety, especially C1′, C2′ and C3′ was observed. As C4′ and C5′ showed none or only weak contributions to all dNTP binding due to the greater distance to the protein surface and therefore a significant contribution of the triphosphate moiety is unlikely. Further bind- ing contributions of the dNTPs to VPg are located in the nucleobase moieties, mainly the C8 of adenine and guanine and the C5 for the pyrimidine nucleobases. Interestingly, for dATP the binding the STD effect the most important moiety for binding interactions, compared to the analogue purine base dGTP in which the highest binding contribution is based in the deoxyribose moiety. Because (c) dGTP and (d) dUTP. The ligand signals are indicated by roman numbers. Relative h spectrum. The CH hydrogens from deoxyribose are associated to 100% STD effect of VPg with dATP has greater specificity than to the other dNTPs. http://bioportal.weizmann.ac.il/fldbin/finde http://bioportal.weizmann.ac.il/fldbin/finde http://bioportal.weizmann.ac.il/fldbin/finde http://bioportal.weizmann.ac.il/fldbin/finde http://bioportal.weizmann.ac.il/fldbin/finde http://bioportal.weizmann.ac.il/fldbin/finde http://bioportal.weizmann.ac.il/fldbin/finde 1 f Biolo d i t O i o N e i h o f ( e g d s p a i i w s i c a t A I M A [ [ [ [ [ [ [ [ [ [ [ [ [ [ [24] M. Mayer, B. Meyer, J. Am. Chem. Soc. 123 (2001) 6108–6117. [25] M. Pellecchia, I. Bertini, D. Cowburn, C. Dalvit, E. Giralt, W. Jahnke, T.L. James, 84 R.B. Mariutti et al. / International Journal o ATP uses the purine motif as the main driving force for bind- ng, we hypothesize that molecular recognition is more specific for his nucleotide. These results are in agreement with the results of lspert et al. [11] who characterized interactions between threon- ne of VPg and nucleotides. Also, to further assess the importance f the deoxyribose moiety in the VPg and dATP interaction, STD- MR experiments were conducted for adenine and a competition xperiment between dATP and adenine. Results indicate that the nteraction between VPg and adenine still takes place and that VPg as higher affinity towards dATP than towards adenine. To address the observed effect of the conformational changes as bserved by CD spectroscopy, we performed STD NMR experiments or dATP under identical conditions but with the addition of 30% v/v) TFE and we observed the same pattern as in Fig. 8. Proteins interact with DNA and RNA via forces which include lectrostatic interactions (salt bridges), dipolar interactions (hydro- en bonding), entropic effects (hydrophobic interactions) and ispersion forces (base stacking) whereby this interaction is ignificantly influenced by their tertiary structures. Beside the hosphodiester bonds, other interactions between nucleic acids nd VPg of SBMV may exist and this is not affected by the changes n the percentage of secondary structure. The addition of dATP n VPg solutions does not change the fold of protein as observed ith TFE, but results in a decrease of the hydrodynamic radius, uggesting that the binding of this ligand reduces intermolecular nteractions. Both the computational and experimental data indi- ate the possibility that the structure of VPg, especially the central nd C-terminal regions, undergo a conformational change when he local hydrophobicity changes. uthor contributions R.B. Mariutti and A. Ullah expressed and purified the protein. .P. Caruso performed CD and fluorescence measurements. F.R. De orais and D. Rehders performed the NMR experiment and R.K. rni analyzed the data. [ gical Macromolecules 83 (2016) 178–184 Acknowledgments This research was supported by grants from PROPe-UNESP, CNPq, FAPESP and CAPES (Brazil). 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http://refhub.elsevier.com/S0141-8130(15)30118-5/sbref0260 http://refhub.elsevier.com/S0141-8130(15)30118-5/sbref0260 http://refhub.elsevier.com/S0141-8130(15)30118-5/sbref0260 http://refhub.elsevier.com/S0141-8130(15)30118-5/sbref0260 http://refhub.elsevier.com/S0141-8130(15)30118-5/sbref0260 http://refhub.elsevier.com/S0141-8130(15)30118-5/sbref0260 http://refhub.elsevier.com/S0141-8130(15)30118-5/sbref0260 Functional expression, monodispersity and conformational changes in the SBMV virus viral VPg on binding TFE 1 Introduction 2 Methods 2.1 Virus purification and RNA extraction 2.2 Primer design, cDNA syntheses and PCR amplification of the ORF1 2.3 Cloning in pGEM-T easy vector 2.4 Cloning in pET28a 2.5 Expression of recombinant VPg in E. coli using pET28a vector 2.6 Protein purification 2.6.1 Purification of the VPg 6xHis recombinant protein 2.7 Dynamic light scattering 2.8 Circular dichroism spectroscopy 2.9 Binding of probe 8-aniline-1-naphthalene sulfonate (ANS) 2.10 Prediction of intrinsic disorder in VPg 2.11 Saturation transfer difference nuclear magnetic resonance spectroscopy 3 Results and discussion 3.1 Virus purification, RNA extraction and cloning of VPg of SBMV 3.2 Purification and hexahistidine tag removal 3.3 Dynamic light scattering 3.4 Circular dichroism 3.5 Binding of probe ANS 3.6 Prediction of intrinsically unstructured/disordered sequence 3.7 Nuclear magnetic resonance spectroscopy Author contributions Acknowledgments References