The secondary structure of recombinant proteins can change through complex formation

with other proteins. Here, we have determined the spatial structure of two proteins,

including core protein of hepatitis C virus and HbsAg of hepatitis B virus, without the

effect of human HSP90 as well as with the effect of this recombinant chaperone. As a

result, the increase in intensity from 297.5 to 346.64 was accompanied by different folding

and being non-polar protein in complex with the chaperone. HbsAg protein, combined

with HSP90, showed a reduction in the maximum peak wavelength from 385 to 369.07

nm. The property of protein of being non-polar and hydrophobic, as well as having an

increase in intensity from 200 to 219, indicates the protein folding. The shift from 342 to

337 nm along with blue shift indicates hydrophobic properties and the removal of protein

from the water environment

Bandehpour, Mojgan.

Yaghoobi, Hajar.

The secondary structure of recombinant proteins can change through complex formation with other proteins. Here, we have determined the spatial structure of two proteins, including core protein of hepatitis C virus and HbsAg of hepatitis B virus, without the effect of human HSP90 as well as with the effect of this recombinant chaperone. As a result, the increase in intensity from 297.5 to 346.64 was accompanied by different folding and being non-polar protein in complex with the chaperone. HbsAg protein, combined with HSP90, showed a reduction in the maximum peak wavelength from 385 to 369.07 nm. The property of protein of being non-polar and hydrophobic, as well as having an increase in intensity from 200 to 219, indicates the protein folding. The shift from 342 to 337 nm along with blue shift indicates hydrophobic properties and the removal of protein from the water environment.Highlights:The structure of recombinant protein is very important in vaccine complex with an adjuvant.The immunity of HCV core and HbsAg proteins could increase in complex with HSP90.Fluorescence spectroscopy and circular dichroism were used to determine the proteins structure. Structural data confirmed the hydrophobic properties if proteins changed in complexation with HSP90

Yaghobi, Hajar

Bandehpour, Mojgan

Journals Portal, Shahid Beheshti University of Medical Sciences

Hajar Yaghoobi a and Mojgan Bandehpour b*a Molecular& Cellular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.b Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.Keywords: Core proteinHbsAgCircular dichroismHIGHLIGHTS•	 The structure of recombinant protein is very important in vaccine complex with an adjuvant.•	 The immunity of HCV core and HbsAg proteins could increase in complex with HSP90.•	 Fluorescence spectroscopy and circular dichroism were used to determine the proteins structure.•	 	Structural	data	confirmed	the	hydrophobic	properties	if	proteins	changed	in	complexation	with	HSP90.* Corresponding Author: Email: m.bandehpour@sbmu.ac.ir  (M. Bandehpour)ABSTRACTThe secondary structure of recombinant proteins can change through complex formation with other proteins. Here, we have determined the spatial structure of two proteins, including core protein of hepatitis C virus and HbsAg of hepatitis B virus, without the effect of human HSP90 as well as with the effect of this recombinant chaperone. As a result, the increase in intensity from 297.5 to 346.64 was accompanied by different folding and being non-polar protein in complex with the chaperone. HbsAg protein, combined with HSP90, showed a reduction in the maximum peak wavelength from 385 to 369.07 nm. The property of protein of being non-polar and hydrophobic, as well as having an increase in intensity from 200 to 219, indicates the protein folding. The shift from 342 to 337 nm along with blue shift indicates hydrophobic properties and the removal of protein from the water environment.Effect of Human HSP90 on Secondary and Tertiary Structures of Core Protein of Hepatitis C Virus and HbsAg of Hepatitis B VirusOriginal Research ArticleTrends in Peptide and Protein Sciences2016ˏ Vol. 1ˏ  No.2ˏ68-72Article history:Received: 2 July 2016Accepted: 13 November 2016Introduction The structure of the recombinant protein is always very important in vaccine complex with an adjuvant. In our previous project (Bandehpour et al., 2008), we have reached the increased immunity of HCV core and HbsAg proteins in complex with HSP90. HBV is a member of the hepadnavirus family prototype. The membrane proteins of HBV are encoded by the ORF S and located within lipid bilayer membrane of the virus. These proteins are involved in the binding to the receptors, the assembly of virus, and secretion. Three main proteins, L, M and S, were produced from one of the three genes, including pre-S1, pre-S2 and S (Liang, 2009). Six genotypes of HBV were divided into 9 subtypes from A to F in accordance with antigenic indexes of protein S. There was a difference of up to 4% between them. Hepatitis C is very similar to pestiviruses and flaviviruses with respect to the genomic and	 hydrophobicity	 profiles	 of	 polypeptides.	 This	 virus	was divided into a separate strain from the Flaviviridae family. Hepatitis C has positive single-stranded RNA with 9,500	nucleotides.	The	first	191	amino	acids	formed	the	core protein with molecular weight 19 to 23 kDa (Knipe and Howley, 2013). At the same time, this protein entered the endoplasmic reticulum, and it was translated, and its signal peptide was isolated by peptidase located in the membrane and the mature form of core protein (179 amino acids) was released into the cytoplasm (Buratti et al., 1997).In	 the	 present	 study,	 fluorescence	 spectroscopy	 and	circular dichroism were used to determine the spatial structure of these two proteins, including core protein of hepatitis C virus and HbsAg of hepatitis B virus without the effect of human HSP90 as well as with the effect of this recombinant chaperone. Materials and MethodsMaterials Core protein of hepatitis C virus, HbsAg of hepatitis B virus and the human HSP90 were previously expressed recombinantly	and	purified	in	our	lab	(Bandehpour	et	al.,	2008). All other chemicals were supplied from Merck Chemical Company (Germany).Determination of secondary and tertiary structures of core protein and HbsAgThe	purified	core	and	HbsAg	proteins	were	investigated	using Circular Dichroism Spectrometer (Model 215, company Aviv, USA). A few steps of dialysis with PBS, containing 4 M, 2 M, and 0.5 M urea, were used to remove urea	 from	purified	protein.	Tm determination of core protein and HbsAgThe folding of the protein structure was disturbed to study the chaperone effect of HSP90 on the proteins studied. For this purpose, heat was used. Tm is the temperature at which half the interest protein is deformed. Therefore, each of the proteins, core and HbsAg, was placed at temperatures 4 °C to 90 °C, and protein absorption was determined in the wavelength 280 nm with an interval of 5 degrees by spectrophotometer. The curve obtained from the temperature and absorbance (OD) was drawn for each protein. The Tm of protein is 4 to 5 degrees lower than the temperature at which the curve of protein is raised (Crowther	et	al.,	2009).	At	first,	200	μg/ml	of	each	protein	was brought to the temperature of unfolding. Then, it was mixed	with	10	μg/ml	of	HSP90,	and	incubated	at	37	°C	for	 an	 hour.	 The	 affinity	 chromatography	 was	 used	 to	remove HSP90 from this mixture. Study of protein secondary structure with circular dichroism (CD)The secondary structure of proteins was determined in the region of Far-UV spectra with a range wavelength 190 to 250 nanometres. With this technique, the percentage of alpha helix, beta sheet and irregular structures (random coil) were determined in the totality of the molecule. 200 µl	of	each	protein,	with	a	concentration	of	200	μg	/ml	in	PBS buffer, was used. Study of third structure with CDThe near-UV spectrum (e.g. 250 to 350 nm) was used to study protein tertiary structure. For this purpose, some 400	μl	protein,	with	 the	concentration	of	1	mg/ml	seen,	was examined. Data obtained from the study of protein structure using Circular Dichroism Spectrometer, Model 215, was analysed using the software CDNN (Circular Dichroism Nerve Network).Fluorescence spectroscopyFluorescence spectroscopy was performed using a fluorometer (Jenway 62 series). The amino acids containing the cyclic substituent, such as tryptophan, tyrosine and phenylalanine,	 had	 the	 property	 of	 fluorescence.	 These	amino acids were excited in the wavelengths 260 to 300 nm, and they emitted a wavelength of 300 to 400 nm. For this	purpose,	600µl	of	protein	with	concentration	1	mg/ml	rose. The excitation wavelength was entirely in 280 nm, and the emission on the main in wavelengths 300 to 400 nm. According to information obtained by this method, the charts of each protein were stable, using Microsoft Office	Excel	2007.ResultsStudy of secondary structure of core and HbsAg proteins by CDAs	observed	in	Fig.	1,	the	chart	identified	by	brown	colour	was the chart of alpha helix for core protein after exposure to HSP90 in vitro. A maximum and two minima were observed in the wavelength range of 200 nm and the range of 220 to 225 nm respectively (Fig. 1).Protein HbsAg (blue graph) and the others reviewed in the chart obtained from far UV did not show any particular structure. This might be the result of several forms of the protein or the being as the membrane protein. The types of oligomers (electrophoresis of protein in non-denaturation condition) were so caused so that the suitable range of this protein did not obtain the secondary structure of core protein. However, the alpha form of this protein was achieved in the mixed form with chaperone and	 separation	 with	 affinity	 chromatography.Data obtained from the study of proteins with near UV were studied with software CDNN. In the spectrum obtained	from	the	core	protein	and	core	/HSP,	the	signals	detected showed the change of waving protein and the position change of aromatic amino acids in protein structure. No signal was observed for HbsAg protein in the mixed form with HSP. Therefore, HSP did not have Effect of human HSP90 on core and HbsAg proteins structure69H. Yaghoobi & M. Bandehpour / TPPS 2016 1(2) 68-7270the effect on correct folding of the protein (Fig. 1).Percentages of α-helix, β sheet, turn, and random coil in proteins structure of core and HbsAgThe alpha helix showed 68.7% and 43.47% decrease in the secondary fabric of both proteins, core and HbsAg, in the mixed form with HSP90. About 20% increase was reported in beta-sheet structures. When the expression of HSP90 proteins core and HbsAg occur in at the same time of chaperone HSP90, the 29.4% and 34.8% decreases were observed in the alpha structure. In contrast, 55% and 20% increases were found in the beta structure. Random and third	structures	did	not	show	significant	changes	(Table	1).Fluorescence study of the structures of proteins core and core + HSP90 The	 proteins	 had	 three	 amino	 acids	 with	 fluorescent	characteristics. They were tyrosine, phenylalanine, and tryptophan. The highest emission was observed in tryptophan. The absorption wavelength of this amino acid was higher than other amino acids. The energy released by phenylalanine and tyrosine often transferred to the tryptophan in the same protein. The excitation and emission	 of	 these	 proteins	were	 studied	 by	 fluorometer	respectively in the wavelengths of 280 nm and the range of 300–400 nm.As observed in Fig. 2, drawn from the results obtained by	 Eclipse	 software,	 the	 decrease	 in	 wavelength	 (blue	shift) in the tryptophan range was seen from 342 to 337 nm, as the pure form of core protein was compared with this protein in the mixed form with the chaperone. This result was so represented that this protein in the mixed form with chaperone had more hydrophobicity than the previous form. The intensity also increased from 297.5 to 346.64, indicating the folding change of the protein in the mixed form with the chaperone (Fig. 2).Fluorescence study of the protein structures of HbsAg and HbsAg + HSP90 According to the graph obtained from the study of HbsAg protein	 with	 the	 mixed	 form	 with	 HSP90	 by	 Eclipse	software, the decrease in wavelength was observed from 385 to 369.07 nm or blue shift in the chart of tryptophan. Non-polar and hydrophobicity of protein, as well as the increase in the intensity of the 200.021 to 219.581, indicated that there were changes in the folding of the protein (Fig. 3).DiscussionHSP90 chaperones are used for the stability and activity of a diverse range of proteins (Zuehlke and Johnson, 2010). HSP90 had the monomer and homodimer states at 37 °C. In this condition, it showed a little tendency of binding to peptides. By raising the temperature to 60 °C, it formed	oligomers	and	their	binding	affinity	was	increased	(Thorne and McQuade, 2004). In this study, HSP90 was evaluated	by	electrophoresis	on	SDS-PAGE	gel	at	37	°C.	As	found	in	a	study	(Nakai	et	al.,	2006),	protein	purified	by	affinity	chromatography	formed	multimeric	states.	In	this study, 191 amino acids of this protein were used. It is known	that	 the	 total	sequence	of	 this	protein	 influenced	the formation of its spatial structure. Owing to S and poly-histidine	 peptides,	 affinity	 chromatography	was	 used	 to	purify proteins core and HbsAg. Poly-histidine sequences had the highest application in the preparation and purification	of	recombinant	proteins.	Doody	et	al.	(2004)	found that glycoprotein 96 is capable of forming twisting in MHC I and MHC II proteins and plays a role in antigen presentation. Kunkel and Watowich have investigated the biophysical characterization of the core protein of HCV virus. They found that this protein was present as Table 1. Percentage of secondary structure in proteins Core and HbsAg.           structure α-Helix(%)β-Sheet(%)Beta-turn(%)Random coil(%)Total sum(%)proteinCore 15 31.2 18.1 45.2 109.5Core + HSP 1 10.3 37.8 14.7 39.4 102.1Core	/	HSP	2 4.4 56.5 17.1 32.3 110.3HbsAg 13.8 50.2 21.6 32.1 117.7HbsAg + HSP 3 6 59.8 15.3 29.9 110.9HbsAg	/	HSP	4 4.8 59.2 18.9 28.6 111.61 The core protein was combined with chaperon in vitro.2 The core protein was expressed at the same time of chaperon.3 HbsAg protein was combined with chaperon in vitro.4 HbsAg protein was expressed at the same time of chaperon.71Effect of human HSP90 on core and HbsAg proteins structure13   Figure 1               Figure 1. Near UV CD spectra of proteins Hbs and core in phosphate buffer pH 7.4.Figure 2. Fluorescence emission spectra of pure core protein (red) and the protein mixed with chaperon with ratio 5% (blue).14   Figure 2           Figure 3. Fluorescence emission spectra of pure HbsAg protein (blue) and the protein mixed with chaperon with 5% ratio (red).15    Figure 3       H. Yaghoobi & M. Bandehpour / TPPS 2016 1(2) 68-7272large multimers in physiological conditions. The study by Far-CD showed that this protein had the strong secondary structure (Kunkel and Watowich, 2004). Our review also indicates that the noise on charts Far-CD	 was	 consistent	 with	 this	 subject.	 The	 fluorescence	spectroscopy and near-UV showed that the regions rich in tryptophan (amino acids 76 to 113) were ultimately located on the surface of the protein, and they were in contact with the solution. The curve obtained from near-UV of core protein showed a signal for tyrosine (285 nm). The signals of tyrosine and tryptophan(290–300nm) were observed	 in	 the	curve	of	core/HSP,	 indicating	 that	 these	proteins folded and the aromatic amino acids exposed to the solution. Regression analyses of the CD in protein secondary structure showed that there were 16% alpha helix, 29% beta-sheets as well as turns and 55% random twists in the studied protein. The results obtained from this study were consistent with our results. There were 15% alpha helix, 33.2% beta-sheets and 18.1% turns and 45.2% random twists. Some percentage of differences may be the result of changes in the sequence. Li et al. found that the heat, ions, vertex and adsorption increased the hydrophobic levels and aggregation of HbsAg protein. After	these	changes,	the	percentage	of	α-helix	decreased	from	48.2%	 to	34.4%	and	γ-turn	 increased	 from	29.6%	to 38.7% (Li et al., 2007). We found that the secondary structure of proteins core and HbsAg, combined with HSP90, showed 68.7% and 43.47% reductions in alpha-helix structure. However, when they expressed at the same time, 29.4% and 34.8% reductions were observed in alpha helix structure. They showed 55% and 20% increases in beta-sheet structures. Random structures	 and	 turns	 show	 no	 significant	 changes.	 The	comparison ofthe optimal effect of chaperones on protein structure indicated that the aromatic amino acids like tryptophan showed the reduction in the peak of fluorescence	 graph	 in	 pure	 form.	The	 shift	 from	342	 to	337 nm along with blue shift caused the protein to show hydrophobic properties and removed from the water environment. As a result, the increase of intensity from 297.5 to346.64 was accompanied with folding and being non-polar of protein in form mixed with a chaperone. HbsAg protein, combined with HSP, showed the reduction of peak wavelength from 385 to 369.07 nm. The non-polar and hydrophobic properties of protein, as well as the increase in intensity from 200 to 219, indicated the protein folding. The shift from 342 to 337 nm along with blue shift caused that the protein showed hydrophobic properties when removed from the water environment.ConclusionIn the present study, the HSP90 that was introduced as adjuvant	in	vaccine	formulation	has	a	significant	effect	on	core protein of hepatitis C virus and HbsAg of hepatitis B virus structures. AcknowledgementsWe thank the Vice-Chancellor of Research, Shahid Beheshti University of Medical Sciences.Competing Interests The authors declared that there are no competing interests. ReferencesBandehpour, M., Khodabandeh, M. and B. Kazemi, (2008)."Cloning and	 Expression	 of	 Hepatitis	 B	 Surface	Antigen."	Hepatitis Monthly, 2008(1, Winter), 17-21. Buratti,	E.,	Di	Michele,	M.,	Song,	P.,	Monti-Bragadin,	C.,	Scodeller,	E.,	 Baralle,	 F.	 and	 S.	 Tisminetzky,	 (1997)."	 Improved	 reactivity	 of	hepatitis C virus core protein epitopes in a conformational antigen-presenting system. "Clinical and Diagnostic Laboratory Immunology, 4(2), 117-121. Crowther, G. J., Napuli, A. J., Thomas, A. P., Chung, D. J., Kovzun, K. V., Leibly, D. J., Castaneda, L. J., Bhandari, J., Damman, C. J., Hui, R. and W. G.  Hol, (2009)." Buffer optimization of thermal melt assays of Plasmodium proteins for detection of small-molecule ligands." Journal of Biomolecular Screening, 14(6), 700-707. Knipe, D. M. and P. Howley, (2013). " Fields Virologym, " 6th ed., Vol. 1, Philadelphia, Wolters Kluwer, Lippincott Williams & Wilkins, pp. 712-824.Kunkel, M. and S. J. Watowich, (2004)." Biophysical characterization of hepatitis C virus core protein: implications for interactions within the virus and host." FEBS Letters, 557(1-3), 174-180. Li, Y., Bi, J., Zhou, W., Huang, Y., Sun, L., Zeng, A. P., Ma, G. and Z. Su, (2007)." Characterization of the large size aggregation of hepatitis B virus	surface	antigen	(HBsAg)	formed	in	ultrafiltration	process."Process Biochemistry, 42(3), 315-319. Liang, T. J. (2009)." Hepatitis B: the virus and disease." Hepatology, 49(S5), S13-S21.Nakai, K., Okamoto, T., Kimura-Someya, T., Ishii, K., Lim, C. K., Tani,	H.,	Matsuo,	E.,	Abe,	T.,	Mori,	Y.,	Suzuki,	T.	 and	T.	Miyamura,	(2006)." Oligomerization of hepatitis C virus core protein is crucial for interaction	with	the	cytoplasmic	domain	of	E1	envelope	protein."Journal of Virology, 80(22), 11265-11273. Thorne,	 M.	 E.	 and	 K.	 L.	 McQuade,	 (2004)."	 Heat-induced	oligomerization of gp96 occurs via a site distinct from substrate binding and is regulated by ATP." Biochemical and Biophysical Research Communications, 323(4), 1163-1171. Zuehlke, A. and    J. L. Johnson, (2010)." HSP90    and      co-chaperones    twist  the functions of diverse client proteins." Biopolymers, 93(3), 211-217.

English

Effect of Human HSP90 on Secondary and Tertiary Structures of Core Protein of Hepatitis C Virus and HbsAg of Hepatitis B Virus

shahrekord university of medical scinces

Hajar Yaghoobi a and Mojgan Bandehpour b*a Molecular& Cellular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.b Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.Keywords: Core proteinHbsAgCircular dichroismHIGHLIGHTS•	 The structure of recombinant protein is very important in vaccine complex with an adjuvant.•	 The immunity of HCV core and HbsAg proteins could increase in complex with HSP90.•	 Fluorescence spectroscopy and circular dichroism were used to determine the proteins structure.•	 	Structural	data	confirmed	the	hydrophobic	properties	if	proteins	changed	in	complexation	with	HSP90.* Corresponding Author: Email: m.bandehpour@sbmu.ac.ir  (M. Bandehpour)ABSTRACTThe secondary structure of recombinant proteins can change through complex formation with other proteins. Here, we have determined the spatial structure of two proteins, including core protein of hepatitis C virus and HbsAg of hepatitis B virus, without the effect of human HSP90 as well as with the effect of this recombinant chaperone. As a result, the increase in intensity from 297.5 to 346.64 was accompanied by different folding and being non-polar protein in complex with the chaperone. HbsAg protein, combined with HSP90, showed a reduction in the maximum peak wavelength from 385 to 369.07 nm. The property of protein of being non-polar and hydrophobic, as well as having an increase in intensity from 200 to 219, indicates the protein folding. The shift from 342 to 337 nm along with blue shift indicates hydrophobic properties and the removal of protein from the water environment.Effect of Human HSP90 on Secondary and Tertiary Structures of Core Protein of Hepatitis C Virus and HbsAg of Hepatitis B VirusOriginal Research ArticleTrends in Peptide and Protein Sciences2016ˏ Vol. 1ˏ  No.2ˏ68-72Article history:Received: 2 July 2016Accepted: 13 November 2016Introduction The structure of the recombinant protein is always very important in vaccine complex with an adjuvant. In our previous project (Bandehpour et al., 2008), we have reached the increased immunity of HCV core and HbsAg proteins in complex with HSP90. HBV is a member of the hepadnavirus family prototype. The membrane proteins of HBV are encoded by the ORF S and located within lipid bilayer membrane of the virus. These proteins are involved in the binding to the receptors, the assembly of virus, and secretion. Three main proteins, L, M and S, were produced from one of the three genes, including pre-S1, pre-S2 and S (Liang, 2009). Six genotypes of HBV were divided into 9 subtypes from A to F in accordance with antigenic indexes of protein S. There was a difference of up to 4% between them. Hepatitis C is very similar to pestiviruses and flaviviruses with respect to the genomic and	 hydrophobicity	 profiles	 of	 polypeptides.	 This	 virus	was divided into a separate strain from the Flaviviridae family. Hepatitis C has positive single-stranded RNA with 9,500	nucleotides.	The	first	191	amino	acids	formed	the	core protein with molecular weight 19 to 23 kDa (Knipe and Howley, 2013). At the same time, this protein entered the endoplasmic reticulum, and it was translated, and its signal peptide was isolated by peptidase located in the membrane and the mature form of core protein (179 amino acids) was released into the cytoplasm (Buratti et al., 1997).In	 the	 present	 study,	 fluorescence	 spectroscopy	 and	circular dichroism were used to determine the spatial structure of these two proteins, including core protein of hepatitis C virus and HbsAg of hepatitis B virus without the effect of human HSP90 as well as with the effect of this recombinant chaperone. Materials and MethodsMaterials Core protein of hepatitis C virus, HbsAg of hepatitis B virus and the human HSP90 were previously expressed recombinantly	and	purified	in	our	lab	(Bandehpour	et	al.,	2008). All other chemicals were supplied from Merck Chemical Company (Germany).Determination of secondary and tertiary structures of core protein and HbsAgThe	purified	core	and	HbsAg	proteins	were	investigated	using Circular Dichroism Spectrometer (Model 215, company Aviv, USA). A few steps of dialysis with PBS, containing 4 M, 2 M, and 0.5 M urea, were used to remove urea	 from	purified	protein.	Tm determination of core protein and HbsAgThe folding of the protein structure was disturbed to study the chaperone effect of HSP90 on the proteins studied. For this purpose, heat was used. Tm is the temperature at which half the interest protein is deformed. Therefore, each of the proteins, core and HbsAg, was placed at temperatures 4 °C to 90 °C, and protein absorption was determined in the wavelength 280 nm with an interval of 5 degrees by spectrophotometer. The curve obtained from the temperature and absorbance (OD) was drawn for each protein. The Tm of protein is 4 to 5 degrees lower than the temperature at which the curve of protein is raised (Crowther	et	al.,	2009).	At	first,	200	μg/ml	of	each	protein	was brought to the temperature of unfolding. Then, it was mixed	with	10	μg/ml	of	HSP90,	and	incubated	at	37	°C	for	 an	 hour.	 The	 affinity	 chromatography	 was	 used	 to	remove HSP90 from this mixture. Study of protein secondary structure with circular dichroism (CD)The secondary structure of proteins was determined in the region of Far-UV spectra with a range wavelength 190 to 250 nanometres. With this technique, the percentage of alpha helix, beta sheet and irregular structures (random coil) were determined in the totality of the molecule. 200 µl	of	each	protein,	with	a	concentration	of	200	μg	/ml	in	PBS buffer, was used. Study of third structure with CDThe near-UV spectrum (e.g. 250 to 350 nm) was used to study protein tertiary structure. For this purpose, some 400	μl	protein,	with	 the	concentration	of	1	mg/ml	seen,	was examined. Data obtained from the study of protein structure using Circular Dichroism Spectrometer, Model 215, was analysed using the software CDNN (Circular Dichroism Nerve Network).Fluorescence spectroscopyFluorescence spectroscopy was performed using a fluorometer (Jenway 62 series). The amino acids containing the cyclic substituent, such as tryptophan, tyrosine and phenylalanine,	 had	 the	 property	 of	 fluorescence.	 These	amino acids were excited in the wavelengths 260 to 300 nm, and they emitted a wavelength of 300 to 400 nm. For this	purpose,	600µl	of	protein	with	concentration	1	mg/ml	rose. The excitation wavelength was entirely in 280 nm, and the emission on the main in wavelengths 300 to 400 nm. According to information obtained by this method, the charts of each protein were stable, using Microsoft Office	Excel	2007.ResultsStudy of secondary structure of core and HbsAg proteins by CDAs	observed	in	Fig.	1,	the	chart	identified	by	brown	colour	was the chart of alpha helix for core protein after exposure to HSP90 in vitro. A maximum and two minima were observed in the wavelength range of 200 nm and the range of 220 to 225 nm respectively (Fig. 1).Protein HbsAg (blue graph) and the others reviewed in the chart obtained from far UV did not show any particular structure. This might be the result of several forms of the protein or the being as the membrane protein. The types of oligomers (electrophoresis of protein in non-denaturation condition) were so caused so that the suitable range of this protein did not obtain the secondary structure of core protein. However, the alpha form of this protein was achieved in the mixed form with chaperone and	 separation	 with	 affinity	 chromatography.Data obtained from the study of proteins with near UV were studied with software CDNN. In the spectrum obtained	from	the	core	protein	and	core	/HSP,	the	signals	detected showed the change of waving protein and the position change of aromatic amino acids in protein structure. No signal was observed for HbsAg protein in the mixed form with HSP. Therefore, HSP did not have Effect of human HSP90 on core and HbsAg proteins structure69H. Yaghoobi & M. Bandehpour / TPPS 2016 1(2) 68-7270the effect on correct folding of the protein (Fig. 1).Percentages of α-helix, β sheet, turn, and random coil in proteins structure of core and HbsAgThe alpha helix showed 68.7% and 43.47% decrease in the secondary fabric of both proteins, core and HbsAg, in the mixed form with HSP90. About 20% increase was reported in beta-sheet structures. When the expression of HSP90 proteins core and HbsAg occur in at the same time of chaperone HSP90, the 29.4% and 34.8% decreases were observed in the alpha structure. In contrast, 55% and 20% increases were found in the beta structure. Random and third	structures	did	not	show	significant	changes	(Table	1).Fluorescence study of the structures of proteins core and core + HSP90 The	 proteins	 had	 three	 amino	 acids	 with	 fluorescent	characteristics. They were tyrosine, phenylalanine, and tryptophan. The highest emission was observed in tryptophan. The absorption wavelength of this amino acid was higher than other amino acids. The energy released by phenylalanine and tyrosine often transferred to the tryptophan in the same protein. The excitation and emission	 of	 these	 proteins	were	 studied	 by	 fluorometer	respectively in the wavelengths of 280 nm and the range of 300–400 nm.As observed in Fig. 2, drawn from the results obtained by	 Eclipse	 software,	 the	 decrease	 in	 wavelength	 (blue	shift) in the tryptophan range was seen from 342 to 337 nm, as the pure form of core protein was compared with this protein in the mixed form with the chaperone. This result was so represented that this protein in the mixed form with chaperone had more hydrophobicity than the previous form. The intensity also increased from 297.5 to 346.64, indicating the folding change of the protein in the mixed form with the chaperone (Fig. 2).Fluorescence study of the protein structures of HbsAg and HbsAg + HSP90 According to the graph obtained from the study of HbsAg protein	 with	 the	 mixed	 form	 with	 HSP90	 by	 Eclipse	software, the decrease in wavelength was observed from 385 to 369.07 nm or blue shift in the chart of tryptophan. Non-polar and hydrophobicity of protein, as well as the increase in the intensity of the 200.021 to 219.581, indicated that there were changes in the folding of the protein (Fig. 3).DiscussionHSP90 chaperones are used for the stability and activity of a diverse range of proteins (Zuehlke and Johnson, 2010). HSP90 had the monomer and homodimer states at 37 °C. In this condition, it showed a little tendency of binding to peptides. By raising the temperature to 60 °C, it formed	oligomers	and	their	binding	affinity	was	increased	(Thorne and McQuade, 2004). In this study, HSP90 was evaluated	by	electrophoresis	on	SDS-PAGE	gel	at	37	°C.	As	found	in	a	study	(Nakai	et	al.,	2006),	protein	purified	by	affinity	chromatography	formed	multimeric	states.	In	this study, 191 amino acids of this protein were used. It is known	that	 the	 total	sequence	of	 this	protein	 influenced	the formation of its spatial structure. Owing to S and poly-histidine	 peptides,	 affinity	 chromatography	was	 used	 to	purify proteins core and HbsAg. Poly-histidine sequences had the highest application in the preparation and purification	of	recombinant	proteins.	Doody	et	al.	(2004)	found that glycoprotein 96 is capable of forming twisting in MHC I and MHC II proteins and plays a role in antigen presentation. Kunkel and Watowich have investigated the biophysical characterization of the core protein of HCV virus. They found that this protein was present as Table 1. Percentage of secondary structure in proteins Core and HbsAg.           structure α-Helix(%)β-Sheet(%)Beta-turn(%)Random coil(%)Total sum(%)proteinCore 15 31.2 18.1 45.2 109.5Core + HSP 1 10.3 37.8 14.7 39.4 102.1Core	/	HSP	2 4.4 56.5 17.1 32.3 110.3HbsAg 13.8 50.2 21.6 32.1 117.7HbsAg + HSP 3 6 59.8 15.3 29.9 110.9HbsAg	/	HSP	4 4.8 59.2 18.9 28.6 111.61 The core protein was combined with chaperon in vitro.2 The core protein was expressed at the same time of chaperon.3 HbsAg protein was combined with chaperon in vitro.4 HbsAg protein was expressed at the same time of chaperon.71Effect of human HSP90 on core and HbsAg proteins structure13   Figure 1               Figure 1. Near UV CD spectra of proteins Hbs and core in phosphate buffer pH 7.4.Figure 2. Fluorescence emission spectra of pure core protein (red) and the protein mixed with chaperon with ratio 5% (blue).14   Figure 2           Figure 3. Fluorescence emission spectra of pure HbsAg protein (blue) and the protein mixed with chaperon with 5% ratio (red).15    Figure 3       H. Yaghoobi & M. Bandehpour / TPPS 2016 1(2) 68-7272large multimers in physiological conditions. The study by Far-CD showed that this protein had the strong secondary structure (Kunkel and Watowich, 2004). Our review also indicates that the noise on charts Far-CD	 was	 consistent	 with	 this	 subject.	 The	 fluorescence	spectroscopy and near-UV showed that the regions rich in tryptophan (amino acids 76 to 113) were ultimately located on the surface of the protein, and they were in contact with the solution. The curve obtained from near-UV of core protein showed a signal for tyrosine (285 nm). The signals of tyrosine and tryptophan(290–300nm) were observed	 in	 the	curve	of	core/HSP,	 indicating	 that	 these	proteins folded and the aromatic amino acids exposed to the solution. Regression analyses of the CD in protein secondary structure showed that there were 16% alpha helix, 29% beta-sheets as well as turns and 55% random twists in the studied protein. The results obtained from this study were consistent with our results. There were 15% alpha helix, 33.2% beta-sheets and 18.1% turns and 45.2% random twists. Some percentage of differences may be the result of changes in the sequence. Li et al. found that the heat, ions, vertex and adsorption increased the hydrophobic levels and aggregation of HbsAg protein. After	these	changes,	the	percentage	of	α-helix	decreased	from	48.2%	 to	34.4%	and	γ-turn	 increased	 from	29.6%	to 38.7% (Li et al., 2007). We found that the secondary structure of proteins core and HbsAg, combined with HSP90, showed 68.7% and 43.47% reductions in alpha-helix structure. However, when they expressed at the same time, 29.4% and 34.8% reductions were observed in alpha helix structure. They showed 55% and 20% increases in beta-sheet structures. Random structures	 and	 turns	 show	 no	 significant	 changes.	 The	comparison ofthe optimal effect of chaperones on protein structure indicated that the aromatic amino acids like tryptophan showed the reduction in the peak of fluorescence	 graph	 in	 pure	 form.	The	 shift	 from	342	 to	337 nm along with blue shift caused the protein to show hydrophobic properties and removed from the water environment. As a result, the increase of intensity from 297.5 to346.64 was accompanied with folding and being non-polar of protein in form mixed with a chaperone. HbsAg protein, combined with HSP, showed the reduction of peak wavelength from 385 to 369.07 nm. The non-polar and hydrophobic properties of protein, as well as the increase in intensity from 200 to 219, indicated the protein folding. The shift from 342 to 337 nm along with blue shift caused that the protein showed hydrophobic properties when removed from the water environment.ConclusionIn the present study, the HSP90 that was introduced as adjuvant	in	vaccine	formulation	has	a	significant	effect	on	core protein of hepatitis C virus and HbsAg of hepatitis B virus structures. AcknowledgementsWe thank the Vice-Chancellor of Research, Shahid Beheshti University of Medical Sciences.Competing Interests The authors declared that there are no competing interests. ReferencesBandehpour, M., Khodabandeh, M. and B. Kazemi, (2008)."Cloning and	 Expression	 of	 Hepatitis	 B	 Surface	Antigen."	 Hepatitis Monthly, 2008(1, Winter), 17-21. Buratti,	E.,	Di	Michele,	M.,	Song,	P.,	Monti-Bragadin,	C.,	Scodeller,	E.,	 Baralle,	 F.	 and	 S.	 Tisminetzky,	 (1997)."	 Improved	 reactivity	 of	hepatitis C virus core protein epitopes in a conformational antigen-presenting system. "Clinical and Diagnostic Laboratory Immunology, 4(2), 117-121. Crowther, G. J., Napuli, A. J., Thomas, A. P., Chung, D. J., Kovzun, K. V., Leibly, D. J., Castaneda, L. J., Bhandari, J., Damman, C. J., Hui, R. and W. G.  Hol, (2009)." Buffer optimization of thermal melt assays of Plasmodium proteins for detection of small-molecule ligands." Journal of Biomolecular Screening, 14(6), 700-707. Knipe, D. M. and P. Howley, (2013). " Fields Virologym, " 6th ed., Vol. 1, Philadelphia, Wolters Kluwer, Lippincott Williams & Wilkins, pp. 712-824.Kunkel, M. and S. J. Watowich, (2004)." Biophysical characterization of hepatitis C virus core protein: implications for interactions within the virus and host." FEBS Letters, 557(1-3), 174-180. Li, Y., Bi, J., Zhou, W., Huang, Y., Sun, L., Zeng, A. P., Ma, G. and Z. Su, (2007)." Characterization of the large size aggregation of hepatitis B virus	surface	antigen	(HBsAg)	formed	in	ultrafiltration	process."Process Biochemistry, 42(3), 315-319. Liang, T. J. (2009)." Hepatitis B: the virus and disease." Hepatology, 49(S5), S13-S21.Nakai, K., Okamoto, T., Kimura-Someya, T., Ishii, K., Lim, C. K., Tani,	H.,	Matsuo,	E.,	Abe,	T.,	Mori,	Y.,	Suzuki,	T.	 and	T.	Miyamura,	(2006)." Oligomerization of hepatitis C virus core protein is crucial for interaction	with	the	cytoplasmic	domain	of	E1	envelope	protein."Journal of Virology, 80(22), 11265-11273. Thorne,	 M.	 E.	 and	 K.	 L.	 McQuade,	 (2004)."	 Heat-induced	oligomerization of gp96 occurs via a site distinct from substrate binding and is regulated by ATP." Biochemical and Biophysical Research Communications, 323(4), 1163-1171. Zuehlke, A. and    J. L. Johnson, (2010)." HSP90    and      co-chaperones    twist  the functions of diverse client proteins." Biopolymers, 93(3), 211-217.

Effect of Human HSP90 on Secondary and Tertiary Structures of Core Protein of Hepatitis C Virus and HbsAg of Hepatitis B Virus

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