Portal de Periódicos da CAPES

Effect of Osmolytes and Chaperone-like Action of P-protein on Folding of Nucleocapsid Protein of Chandipura Virus

2001; Elsevier BV; Volume: 276; Issue: 33 Linguagem: Inglês

10.1074/jbc.m011705200

1083-351X

Amitabha Majumder, Soumen Basak, Tamal Raha, Santanu Pal Chowdhury, Dhrubajyoti Chattopadhyay, Siddhartha Roy,

Animal Virus Infections Studies

Amino acid sequences of nucleocapsid proteins are mostly conserved among different rhabdoviruses. The protein plays a common functional role in different RNA viruses by enwrapping the viral genomic RNA in an RNase-resistant form. Upon expression of the nucleocapsid protein alone in COS cells and in bacteria, it forms large insoluble aggregates. In this work, we have reported for the first time the full-length cloning of the N gene of Chandipura virus and its expression in Escherichia coli in a soluble monomeric form and purification using nonionic detergents. The biological activity of the soluble recombinant protein has been tested, and it was found to possess efficient RNA-binding ability. The state of aggregation of the recombinant protein was monitored using light scattering. In the absence of nonionic detergents, it formed large aggregates. Aggregation was significantly reduced in the presence of osmolytes such asd-sorbitol. Aggregate formation was suppressed in the presence of another viral product, phosphoprotein P, in a chaperone-like manner. Both the osmolyte and phosphoprotein P also suppressed aggregation to a great extent during refolding from a guanidine hydrochloride-denatured form. The function of the phosphoprotein and osmolyte appears to be synergistic to keep the N-protein in a soluble biologically competent form in virus-infected cells. Amino acid sequences of nucleocapsid proteins are mostly conserved among different rhabdoviruses. The protein plays a common functional role in different RNA viruses by enwrapping the viral genomic RNA in an RNase-resistant form. Upon expression of the nucleocapsid protein alone in COS cells and in bacteria, it forms large insoluble aggregates. In this work, we have reported for the first time the full-length cloning of the N gene of Chandipura virus and its expression in Escherichia coli in a soluble monomeric form and purification using nonionic detergents. The biological activity of the soluble recombinant protein has been tested, and it was found to possess efficient RNA-binding ability. The state of aggregation of the recombinant protein was monitored using light scattering. In the absence of nonionic detergents, it formed large aggregates. Aggregation was significantly reduced in the presence of osmolytes such asd-sorbitol. Aggregate formation was suppressed in the presence of another viral product, phosphoprotein P, in a chaperone-like manner. Both the osmolyte and phosphoprotein P also suppressed aggregation to a great extent during refolding from a guanidine hydrochloride-denatured form. The function of the phosphoprotein and osmolyte appears to be synergistic to keep the N-protein in a soluble biologically competent form in virus-infected cells. Chandipura virus vesicular stomatitis virus 5-(2-iodoacetylaminoethyl)naphthalene-1-sulfonic acid 4-morpholinepropanesulfonic acid polymerase chain reaction isopropyl-β-d-thiogalactopyranoside 5,5′-dithiobis(2-nitrobenzoic acid) dynamic light scattering 5,5′-dithiobis(2-nitrobenzoic acid) The (−)-strand RNA viruses (rhabdovirus, influenza, rabies, measles, and Ebola) consist of important human pathogens, including Chandipura virus (CHPV),1 a member of the Rhabdoviridae family (1Bhatt P.N. Rodrigues F.M. J. Med. Res. 1967; 55: 1295-1305Google Scholar). The structure, function, and genetic makeup of this negative sense RNA virus resemble those of vesicular stomatitis virus (VSV). Still, it can be distinguished from two other members of the rhabdovirus family, VSV New Jersey serotype and VSV Indiana serotype, not only in its host species origin and serology (2Dragunova J. Zavada J. Acta Virol. (Prague) (Engl. Ed.). 1979; 23: 319-328PubMed Google Scholar), but also in nucleotide sequences of genes and amino acid sequences of proteins (3Masters P.M. Banerjee A.K. Virology. 1987; 157: 298-306Crossref PubMed Scopus (38) Google Scholar). Upon infection by members of the rhabdovirus family, five major viral proteins, N (nucleocapsid), P (phosphoprotein), L (large), M (matrix), and G (glycoprotein), are synthesized. The N-protein encapsidates genomic RNA in a precise structure that can be compared with histone-mediated enwrapping of a DNA molecule into a nucleosome structure. Only this encapsidated form of the genome can be recognized by viral polymerase as its template during both transcription and replication (4Banerjee A.K. Microbiol. Rev. 1987; 51: 66-87Crossref PubMed Google Scholar). Nucleocapsid proteins not only protect the viral genome from RNase action, but are also thought to play some vital regulatory roles in the transition from transcription to replication in the viral life cycle, referred to as the transcription-replication switch. Previous studies have indicated that the N-protein has a common tendency to form large aggregates that are biologically inactive. In VSV, it was observed that interaction of the N-protein with the P-protein keeps the N-protein in a soluble form in vivo that is capable of enwrapping the de novo synthesized genomic RNA. N-protein/P-protein interaction also may confer specificity for RNA-binding activity (5Masters P.S. Banerjee A.K. J. Virol. 1988; 62: 2658-2664Crossref PubMed Google Scholar) of the N-protein where it channels the N-protein pool to viral RNA sequences only. This regulatory importance of N-protein/P-protein interaction makes it an important biological event in the viral life cycle. In influenza virus, the nucleoprotein was found to interact with viral RNA polymerase, directly modulating its activity (6Biswas S.K. Boutz L.P. Nayak P.D. J. Virol. 1998; 72: 5493-5501Crossref PubMed Google Scholar). In VSV, it has been proposed that the P-protein in its phosphorylated multimeric state forms a complex with the L-protein to produce functional transcriptase, whereas in its unphosphorylated state, it complexes with the L-protein to form replicase. The N-protein has been suggested to be an integral member of the replicase complex (7Pattnaik A.K. Hwang L. Li T. Englund N. Mathur M. Das T. Banerjee A.K. J. Virol. 1997; 71: 8167-8175Crossref PubMed Google Scholar). The multifunctional nature of the N-protein and its interaction with different targets make this viral product an attractive model for a detailed structure-function analysis in the CHPV system so that its precise role in different stages of the viral life cycle can be elucidated. One of the important aspects of N-protein function is the maintenance of its active and soluble form. As mentioned before, in its free form, the protein has a tendency to form aggregates in vitro and perhaps in vivo (8Hsu C.H. Kingsbury D.W. Murti K.G. J. Virol. 1979; 32: 304-313Crossref PubMed Google Scholar). How the cellular environment maintains a soluble and active pool of the N-protein is crucial to understanding the roles played by various cellular factors in the viral life cycle. Clearly, the P-protein increases the solubility of the N-proteinin vivo through formation of complexes. However, the structural nature of the N-protein in these complexes is not known. Thus, the possibility is raised that the P-protein may be interacting with a disordered N-protein in a chaperone-like fashion. Protein folding studies in vivo have elucidated the crucial roles of osmolytes (9Baskakov I. Bolen D.W. J. Biol. Chem. 1988; 273: 4831-4834Abstract Full Text Full Text PDF Scopus (318) Google Scholar) and chaperone systems in suppressing aggregation and shifting the distribution toward the folded state, thereby enhancing the activity and solubility of the proteins. A central point in this study is to understand the roles of the P-protein and intracellular osmolytes in maintaining and enhancing the solubility of the N-protein, thereby ensuring a steady supply for encapsidation of progeny viruses. Pfu DNA polymerase and T4 DNA ligase were from New England Biolabs (Beverly, MA). Tripure reagentTM and Superscript II reverse transcriptase were from Life Technologies, Inc. Klenow polymerase, SP6 RNA polymerase, DNase, 4-nitro blue tetrazolium chloride, and 5-bromo-4-chloro-3-indolyl phosphate were from Roche Molecular Biochemicals. The gel extraction kit was from QIAGEN Inc. The Mono-Q column was from Amersham Pharmacia Biotech (Uppsala, Sweden). Alkaline phosphatase-conjugated goat anti-mouse IgG was from Sigma. I-EDANS was from Molecular Probes, Inc. (Eugene, OR). Prestained molecular mass markers and Bio-Beads SM-2 adsorbent were from Bio-Rad. Other common reagents were of analytical grade. The viral genomic RNA was isolated from CHPV (strain 1653514) using Tripure reagentTMaccording to the manufacturer's protocol. Isolated RNA was estimated qualitatively and quantitatively by running 1% MOPS gel and by spectrophotometric analysis at 260 and 280 nm, respectively. Two terminal primers (DJC1 and DJC2) were designed to clone the N gene by reverse transcription-PCR. Primer DJC1 (5′-TTTATA CATATG AGTTCTCAAGTA-3′) included a hexameric anchor followed by anNdeI restriction enzyme site. The rest of the sequence corresponds to the 5′ terminus of the N gene. In primer DJC2 (5′-TTTATA GGATCC TCATGCAAAGAG-3′), the hexameric anchor was followed by a BamHI restriction enzyme site, and the rest of the sequence was complementary to the 3′-end of the N gene (3Masters P.M. Banerjee A.K. Virology. 1987; 157: 298-306Crossref PubMed Scopus (38) Google Scholar). First-strand cDNA synthesis was carried out with primer DJC1 and viral genomic RNA as template using Superscript II reverse transcriptase, and PCR was done with Pfu polymerase according to the instructions given by the manufacturer. The PCR product was run on a 1% agarose gel in 0.5× Tris borate/EDTA (Fig. 1). The PCR product purified from the 1% agarose gel using QIAGEN gel extraction resin was blunt-ended with Klenow polymerase and cloned intoSmaI-digested pUC18 vector using T4 DNA ligase. The ligation mixture was transformed into Escherichia coli XL1-Blue, and positive clones were confirmed by restriction digestion, followed by direct sequencing of the recombinant DNA (Fig. 1). The full-length N gene of CHPV was digested in the pUC18-NC clone withNdeI and BamHI restriction enzymes, and the released DNA fragment was subcloned intoNdeI/BamHI-cut pET-3a vector under the control of the T7 promoter. The recombinant DNA was digested with different restriction enzymes for further confirmation (Fig. 1). Competent E. coli BL21(DE3) cells were transformed with pET-3a-NC plasmid DNA, and transformed cells were inoculated in 100 ml of Luria broth supplemented with 20 mm glucose containing 100 mg/ml ampicillin and incubated at 37 °C under shaking conditions until A600 = 0.3. Cells were then induced with 500 µm IPTG for 4 h at 37 °C (10Studier F.W. Rosenberg A.H. Dum J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6005) Google Scholar). After harvesting, the cell pellet was suspended in 2 ml of buffer A containing 50 mm Tris-HCl, 1 mm EDTA, and 0.1% Triton X-100. For lysis, lysozyme was added at a concentration of 250 µg/ml to buffer A, and the suspension was kept on ice for 1 h. The cells were then sonicated to reduce the viscosity due to chromosomal DNA, and lysates were clarified by spinning at 13,000 rpm for 30 min at 4 °C. The soluble supernatant was separated from the inclusion body pellet. The inclusion body pellet obtained after centrifugation was denatured and taken into 2 ml of a solution of 8 m urea in buffer A. The soluble and urea fractions were analyzed by 10% SDS-polyacrylamide gel electrophoresis, followed by Coomassie Blue staining. Different parameters such as IPTG concentration, temperature, and duration of induction were varied to maximize expression of the N-protein in soluble form. The N-protein expressed in soluble form was purified from bacterial lysate through a Mono-Q anion-exchange fast protein liquid chromatography column. The column was pre-equilibrated with buffer A containing 200 mm NaCl, and proteins were eluted with a gradient of NaCl from 200 to 700 mm in a total volume of 25 ml of buffer A. The flow rate and fraction size were 0.25 ml/min and 1 ml, respectively. Fractions were analyzed on 10% discontinuous SDS-polyacrylamide gel (acrylamide/bisacrylamide ratio of 30:0.8), followed by Coomassie Blue staining to test the homogeneity of the purified protein. The purified N-protein was stored in buffer A containing 200 mm NaCl and 10% glycerol at −20 °C Proteins were subjected to 10% SDS-polyacrylamide gel electrophoresis, and Western blotting was performed with mouse polyclonal anti-CHPV antibody as the primary antibody and alkaline phosphatase-conjugated goat anti-mouse IgG as the secondary antibody. This was followed by color reaction with 4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate. A 49-nucleotide leader RNA gene of CHVP was cloned under the control of the SP6 promoter in the pGEM-4Z vector. DNA template was linearized with the HindIII restriction enzyme. Radiolabeled positive sense leader RNA was synthesized in vitro in a 40-ml reaction with [α-32P]UTP and SP6 RNA polymerase essentially as described by the manufacturer. DNA template was removed by RQ1 DNase treatment, and the leader RNA was extracted with phenol/chloroform and precipitated twice with ethanol. The product was analyzed on 10% polyacrylamide gel (29:1) containing 8 murea in 1× TAE (40 mm Tris-acetate, 2 mm EDTA, pH 8.0). To study the encapsidation reaction, 32P-labeled leader RNA (200 ng) was incubated with increasing amounts of N-protein (0.3–4.5 µm). The binding reaction was carried out in a total volume of 15 ml in 10 mm Tris-HCl containing 100 mm NaCl, 40 mm KCl, 5 mmMgCl2, 1 mm dithiothreitol, and 5% glycerol at 37 °C for 30 min. In the presence and absence of UV cross-linking (Appligene UV crosslinker), the RNA-protein complex was treated with RNase A at a final concentration of 60 mg/ml for 15 min. The reaction was stopped by the addition of 10× loading dye containing 30% Ficoll, 1 mm EDTA, 0.25% bromphenol blue, and 0.25% xylene cyanol. The reaction mixture was run on a 6% nondenaturing polyacrylamide gel containing 5% glycerol at 4 °C in TAE (40 mm Tris-acetate, 2 mm EDTA, pH 8.0) (acrylamide/bisacrylamide of 30:0.8). The gel was dried and exposed to x-ray film at −70 °C. The P-protein of CHPV was expressed in bacteria, and only the soluble fraction was purified as described earlier (11Raha T. Chattopadhyay D. Chattopadhyay D. Roy S. Biochemistry. 1999; 38: 2110-2116Crossref PubMed Scopus (17) Google Scholar). To determine the number of reactive sulfhydryl groups by DTNB titration, 10 µm CHPV N-protein was incubated with 0.5 mm DTNB in 50 mm Tris-HCl (pH 8) at 25 °C. The reaction was monitored at 412 nm in a Hitachi UV-2000 spectrophotometer after an appropriate base-line correction. A molar extinction coefficient of 1.36 × 104m−1 cm−1 was used to calculate the number of reactive sulfhydryl groups. The purified CHPV N-protein (10 µm) was reacted with 5 mmI-EDANS at 37 °C for 2 h. After incubation, the protein was dialyzed against 50 mm Tris-HCl (pH 8) containing 150 mm NaCl to remove the unreacted I-EDANS. The absorbance at 337 nm was measured to check the incorporation of I-EDANS into the protein with the buffer from the previous dialysis being used for the base line. Circular dichroism spectra were measured in a Jasco J-700 spectropolarimeter. Measurements were carried out in 50 mm Tris-HCl (pH 8) containing 150 mm NaCl at ambient temperature. An average of four scans was taken. A time constant of 2 s and a scan speed of 50 nm/min were used for spectral scanning. The protein concentration was 10 µm. Proper base-line corrections were made. Anisotropy experiments were performed using a Hitachi polarization accessory. The fluorescence intensity componentsI vv, I vh,I hv, and I hh (where the subscripts refer to the horizontal (h) and vertical (v) positioning of the excitation and emission polarizers, respectively) were used to calculate the steady-state fluorescence anisotropy (A) according to the following equation: A = (I vv −GI vh)/(I vv + 2GI vh), where G is the grating factor that corrects for the wavelength-dependent distortions of the polarizing system. Steady-state fluorescence spectra were recorded in a Hitachi F-3010 spectrofluorometer with spectrum addition and subtraction facility. The fluorescence experiments were carried out at 37 °C, and the temperature was maintained by a circulating water bath attached to the spectrofluorometer. The excitation and emission band passes were maintained at 5 nm, and all readings were taken in a cuvette with a 1-cm path length. Light scattering experiments were done to check the state of aggregation of the soluble CHPV N-protein and to monitor the roles of osmolytes and the CHPV P-protein in this aggregation process. The CHPV N-protein at a high concentration was first treated with Bio-Beads SM-2 adsorbent to remove Triton X-100, filtered three times through a Millipore filter, and added to a Millipore filtered buffer under suitable dust-free conditions to a final protein concentration of 50 µm. All additions were made in the cuvette, and light scattering was measured with excitation and emission wavelengths set at 340 nm. To determine the effect of osmolytes, the same experiment was carried out, but a 250 mm concentration of the osmolyte d-sorbitol was added during detergent removal from a high concentration of CHPV N-protein. To determine the effect of the CHPV P-protein on aggregation, the concentrated stock of detergent-removed CHPV N-protein was denatured with 8 m urea. The denatured protein was diluted in a native buffer in the fluorometer cuvette, and scattering intensities were monitored in the presence of varying concentrations of CHPV P-protein and osmolytes. Dynamic light scattering experiments were performed using an Otsuka Electronics DLS700 instrument. For DLS experiments, the concentration of CHPV N-protein was kept at 1 mg/ml, and the concentration of d-sorbitol was kept at 250 mm. In this experiment, the sample was illuminated with a 638.8 helium-neon solid-state laser, and the intensity of light scattered at an angle of 90° was measured. An auto-correlation function was used to determine the translational diffusion coefficient (D T) of the sample particles in the solution by measuring the fluctuations in the intensity of the scattered light. The hydrodynamic radius (R H) of the sample particles was derived from D T using Stokes-Einstein's equation: D T =k B T/6πηR H, where k B is the Boltzmann constant, Tis the absolute temperature in degrees Kelvin, and η is the solvent viscosity. To understand the conformation of the N-protein and its role in the aggregation process, we felt the need to isolate the soluble N-protein, but not a refolded, which may differ in conformation. We observed that when overproduced in bacteria by inducing cells with 500 µm IPTG at 37 °C for 4 h, the N-protein composed more than 50% of the total cellular proteins. However, the majority of the overexpressed protein formed inclusion bodies, posing a problem for its purification (data not shown). We observed that BL21(DE3) cells induced at A 600 = 0.3 with 100 µmIPTG for 14 h at 16 °C produced the majority of the N-protein in a soluble form. To maximize the amount of N-protein in a soluble form during purification, we introduced 200 mm NaCl in buffer A and washed the inclusion pellet several times with the same buffer. We found that the above protocol successfully produces >80% of the overexpressed protein in soluble form (Fig.2, A and B,lane 5). Western blotting was performed to further verify the above results (Fig. 2 B). The major biological role of the nucleocapsid protein is to bind with the viral genome and to encapsidate it in an RNase-resistant form. We tested the affinity of the recombinant protein for viral leader RNA sequence corresponding to the 5′-end of the viral anti-genome by gel electrophoretic mobility shift assays. In vitro transcribed leader RNA was incubated with increasing amounts of recombinant N-protein, and the complex was resolved on 6% native polyacrylamide (see “Experimental Procedures”). We observed the appearance of a shifted band upon the addition of protein, and the intensity of the shifted band increased sharply with increasing protein concentrations, indicating a high affinity of the recombinant protein for leader RNA (Fig. 3 A, firstthrough ninth lanes). We next examined the ability of the recombinant protein to protect viral RNA from RNase action in vitro. To demonstrate that we have treated the complex formed between leader RNA and the N-protein with RNase in another set, the complex was UV-cross-linked and treated with RNase (Fig.3 A, tenth and eleventh lanes). The intensity of the shifted band remained the same as that of the RNase-untreated complex (Fig. 3 A, ninth lane), indicating a true encapsidation of viral leader RNA by the N-protein. CD spectra have been widely used as to monitor secondary structure. Fig.4 shows the far-UV CD spectrum of the solubilized N-protein after removal of Triton X-100. An attempt was made to extract relative amounts of secondary structure present in the protein from the CD spectra. Although the quality of fit was not excellent, it indicated a large amount of random-coil form present. From the work of Baskakov and Bolen (9Baskakov I. Bolen D.W. J. Biol. Chem. 1988; 273: 4831-4834Abstract Full Text Full Text PDF Scopus (318) Google Scholar), it is clear that physiological concentrations of osmolytes can drive the folding equilibrium toward the native state. If the random-coil conformations present in the soluble N-protein reflect partial denaturation, a significant secondary structure alteration of the N-protein is anticipated upon addition of an osmolyte. So, far-UV CD spectra were measured in the presence of an osmolyte (d-sorbitol), and secondary structure formation was monitored. The addition of d-sorbitol led to some enhancement of the CD spectrum and hence secondary structure contents. We also attempted to measure the effect of osmolytes on N-protein structure by the use of steady-state fluorescence anisotropy. Fluorescence anisotropy is a function of probe motion. In the absence of internal probe motions, fluorescence anisotropy is a function of rotational tumbling of the whole molecule. Rapid internal motions, typical of partially or fully disordered states, reduce the anisotropy values. An increase in order generally should increase the fluorescence anisotropy value of a probe covalently attached to the protein. Sulfhydryl groups are selective attachment points for fluorescent probes. The DTNB reaction was used for determining the number of titrable sulfhydryl groups. 2.99 ± 0.1 groups reacted with DTNB, indicating that these sulfhydryl groups are available for attachment of covalent probes. 2.93 ± 0.19 I-EDANS molecules were incorporated into the protein using the protocol described under “Experimental Procedures.” This I-EDANS-labeled N-protein showed full biological activity with respect to RNA-binding ability as shown in Fig. 3 B. Table I reports the steady-state fluorescence anisotropy values of the I-EDANS-labeled soluble recombinant N-protein in the presence and absence of the osmolyted-sorbitol. The anisotropy value in the absence ofd-sorbitol is very low compared with the expected value for a typical globular protein of 48 kDa. For example, when attached to the λ-repressor dimer (molecular mass of 52 kDa), a dansyl chloride probe gave an anisotropy value of ∼0.1 and a rotational correlation time of ∼20 ns under similar conditions (12Banik U. Mandal N.C. Bhattacharya B. Roy S. J. Biol. Chem. 1993; 268: 3938-3943Abstract Full Text PDF PubMed Google Scholar). We measured the lifetimes of the attached I-EDANS probe in the presence and absence ofd-sorbitol. The lifetimes were 15.1 and 15.3 ns in the absence and presence of d-sorbitol, respectively. If one calculates the rotational correlation time from the Perrin equation assuming a limiting anisotropy value of 0.4, the values are 0.8 and 2.2 ns without and with d-sorbitol, respectively. These rotational correlation times clearly indicate that there are very significant internal motions indicative of disorder. The addition ofd-sorbitol led to a significant increase in the fluorescence anisotropy and rotational correlation time, indicating that the addition of osmolytes leads to a decrease in internal motions. This is consistent with the modest enhancement of the CD spectrum in the presence of osmolytes.Table ISteady-state fluorescence anisotropy of the I-EDANS-labeled N protein in the presence and absence of osmolytesd-Sorbitol concFluorescence anisotropy values00.02250 mm0.05A concentrated stock of the soluble recombinant N-protein was labeled with I-EDANS and diluted with 50 mm Tris-HCl (pH 8) containing 100 mm NaCl in the fluorometer cuvette to a final concentration of 0.5 µm. The fluorescence anisotropy of this protein sample was measured in the presence and absence of 250 mmd-sorbitol. Open table in a new tab A concentrated stock of the soluble recombinant N-protein was labeled with I-EDANS and diluted with 50 mm Tris-HCl (pH 8) containing 100 mm NaCl in the fluorometer cuvette to a final concentration of 0.5 µm. The fluorescence anisotropy of this protein sample was measured in the presence and absence of 250 mmd-sorbitol. The spectroscopic studies reported above indicate that the soluble N-protein is partially denatured and disordered. It is generally believed that such partially denatured and disordered proteins are often prone to aggregation. To investigate the self-aggregating tendency of the soluble recombinant N-protein, dynamic light scattering measurements were performed. For this experiment, Triton X-100 was removed, and aggregation was monitored using dynamic light scattering. DLS studies showed that the CHPV N-protein formed large aggregates and that the change in aggregate size occurred with time. While monitoring the aggregation process of the N-protein, it was seen that 15 min after the removal of Triton X-100, the protein started to form aggregates (seen from the increasing value of the Stokes radius). Fig.5 A shows the DLS results after ∼60 min, when the protein formed particles with an average Stokes radius of 8.8 nm (a monomer of the size of the N-protein should have a Stokes radius of ∼2–3 nm). After 18 h, it reached the saturation of the aggregation process and formed particles with an average Stokes radius of 16.05 nm (Fig. 5 B). The standard error of these measurements was ∼5% based on three independent measurements. Clearly, the soluble N-protein has a tendency to aggregate, which is reduced in the presence of nonionic detergents such as Triton X-100. Fluorescence anisotropy indicated that the osmolyted-sorbitol increased the local order in the N-protein. The reduction of disorder, even a local one, may have an influence on the solubility and aggregation of proteins. So we investigated the roles of osmolytes present in the cells in solubilization of the N-protein; and for this purpose, static light scattering experiments were performed. The static light scattering technique is widely used to study protein aggregation and disaggregation (13Bandyopadhyay S. Mukhopadhyay C. Roy S. Biochemistry. 1996; 35: 5033-5044Crossref PubMed Scopus (21) Google Scholar). Aggregation of proteins gives rise to high light scattering values, which reduce upon disaggregation. In this experiment, first Triton X-100 was removed from a very high concentration of soluble recombinant N-protein using Bio-Beads SM-2 adsorbent. The protein was then diluted to a final concentration of 50 µm in a fluorometer cuvette, and scattering values were monitored as a function of time. This same experiment was repeated, butd-sorbitol (250 mm) was included in the buffer during detergent removal and light scattering measurements. TableII reports the light scattering intensities after 30 min of 50 µm N-protein in the presence and absence of osmolytes. The light scattering value was reduced by severalfold in the presence of osmolytes. Clearly, the presence of 250 mmd-sorbitol inhibited aggregation, suggesting that even an increase in the local order leads to inhibition of self-aggregation of the N-protein.Table IIAggregation of Triton X-100-C removed soluble recombinant N-protein in the presence and absence of osmolytes using static light scatteringConditionsScattering intensities50 µm CHPV N-protein876.9750 µm CHPV N-protein + 250 mmd-sorbitol259.3A concentrated stock of the soluble recombinant N-protein was taken and from it was removed Triton X-100 using Bio-Beads SM-2 adsorbent. Then, the protein stock was diluted with 50 mm Tris-HCl (pH 8) containing 100 mm NaCl to a final concentration of 50 µm. Static light scattering intensity was monitored at 340 nm and excited at the same wavelength as a function of time. The same experiment was repeated, but 250 mmd-sorbitol was added during the detergent removal. All values were corrected for the appropriate blank buffer value, and they represent the scattering intensities after 30 min. Open table in a new tab A concentrated stock of the soluble recombinant N-protein was taken and from it was removed Triton X-100 using Bio-Beads SM-2 adsorbent. Then, the protein stock was diluted with 50 mm Tris-HCl (pH 8) containing 100 mm NaCl to a final concentration of 50 µm. Static light scattering intensity was monitored at 340 nm and excited at the same wavelength as a function of time. The same experiment was repeated, but 250 mmd-sorbitol was added during the detergent removal. All values were corrected for the appropriate blank buffer value, and they represent the scattering intensities after 30 min. As seen from the above experiments, the N-protein is prone to aggregation, w