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• W2090161921 abstract "Proteins that belong to the heat shock protein (Hsp) 40 family assist Hsp70 in many cellular functions and are important for maintaining cell viability. A knowledge of the structural and functional characteristics of the Hsp40 family is therefore essential for understanding the role of the Hsp70 chaperone system in cells. In this work, we used small angle x-ray scattering and analytical ultracentrifugation to study two representatives of human Hsp40, namely, DjA1 (Hdj2/dj2/HSDJ/Rdj1) from subfamily A and DjB4 (Hlj1/DnaJW) from subfamily B, and to determine their quaternary structure. We also constructed low resolution models for the structure of DjA1-(1–332), a C-terminal-deleted mutant of DjA1 in which dimer formation is prevented. Our results, together with the current structural information of the Hsp40 C-terminal and J-domains, were used to generate models of the internal structural organization of DjA1 and DjB4. The characteristics of these models indicated that DjA1 and DjB4 were both dimers, but with substantial differences in their quaternary structures: whereas DjA1 consisted of a compact dimer in which the N and C termini of the two monomers faced each other, DjB4 formed a dimer in which only the C termini of the two monomers were in contact. The two proteins also differed in their ability to bind unfolded luciferase. Overall, our results indicate that these representatives of subfamilies A and B of human Hsp40 have different quaternary structures and chaperone functions. Proteins that belong to the heat shock protein (Hsp) 40 family assist Hsp70 in many cellular functions and are important for maintaining cell viability. A knowledge of the structural and functional characteristics of the Hsp40 family is therefore essential for understanding the role of the Hsp70 chaperone system in cells. In this work, we used small angle x-ray scattering and analytical ultracentrifugation to study two representatives of human Hsp40, namely, DjA1 (Hdj2/dj2/HSDJ/Rdj1) from subfamily A and DjB4 (Hlj1/DnaJW) from subfamily B, and to determine their quaternary structure. We also constructed low resolution models for the structure of DjA1-(1–332), a C-terminal-deleted mutant of DjA1 in which dimer formation is prevented. Our results, together with the current structural information of the Hsp40 C-terminal and J-domains, were used to generate models of the internal structural organization of DjA1 and DjB4. The characteristics of these models indicated that DjA1 and DjB4 were both dimers, but with substantial differences in their quaternary structures: whereas DjA1 consisted of a compact dimer in which the N and C termini of the two monomers faced each other, DjB4 formed a dimer in which only the C termini of the two monomers were in contact. The two proteins also differed in their ability to bind unfolded luciferase. Overall, our results indicate that these representatives of subfamilies A and B of human Hsp40 have different quaternary structures and chaperone functions. The Hsp70 1The abbreviations used are: Hsp, heat shock protein; LUC, luciferase; SAXS, small angle X-ray scattering; AUC, analytical ultracentrifugation; G/F-rich, glycine/phenylalanine-rich region; NBD, nucleotide binding domain. chaperone system is formed by Hsp70 (DnaK-related) and its co-chaperones Hsp40 (DnaJ-related) and GrpE. This system assists many cellular processes involving proteins, including folding, transport through membranes, degradation, and escape from aggregation (1.Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3607) Google Scholar, 2.Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2435) Google Scholar, 3.Fink A.L. Physiol. Rev. 1999; 79: 425-449Crossref PubMed Scopus (873) Google Scholar, 4.Mayer M.P. Brehmer D. Gässler C.S. Bukau B. Adv. Protein Chem. 2001; 50: 1-45Google Scholar, 5.Hartl F.U. Hayer-Hartl M. Science. 2002; 295: 1852-1858Crossref PubMed Scopus (2799) Google Scholar, 6.Borges J.C. Ramos C.H.I. Protein Pept. Lett. 2005; 12: 257-261Crossref PubMed Scopus (136) Google Scholar). The affinity of Hsp70 for unfolded proteins is regulated by the binding of either ADP (high affinity) or ATP (low affinity) to its nucleotide binding domain (NBD), and by interaction with its co-chaperones (5.Hartl F.U. Hayer-Hartl M. Science. 2002; 295: 1852-1858Crossref PubMed Scopus (2799) Google Scholar, 7.Mayer M.P. Schröder H. Rüdiger S. Pall K. Laufen T. Bukau B. Nat. Struct. Biol. 2000; 7: 586-593Crossref PubMed Scopus (311) Google Scholar). Hsp40 assists the folding of nascent proteins, and prevents aggregation and the refolding of aggregates by presenting nascent proteins to Hsp70 and stimulating the hydrolysis of ATP (8.Martin J. Hartl F.U. Curr. Opin. Struct. Biol. 1997; 7: 41-52Crossref PubMed Scopus (165) Google Scholar, 9.Hendrick J.P. Langer T. Davis T.A. Hartl F.U. Wiedmann M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10216-10220Crossref PubMed Scopus (145) Google Scholar, 10.Liberek K. Wall D. Georgopoulos C. Proc. Natl. Acad. Sci. U. S. A. 1996; 92: 6224-6228Crossref Scopus (93) Google Scholar, 11.Karzai A.W. MaMacken R. J. Biol. Chem. 1996; 271: 11236-11246Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 12.Laufen T. Mayer M.P. Beisel C. Klostermeier D. Mogk A. Reistein J. Bukau B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5452-5457Crossref PubMed Scopus (475) Google Scholar). Hsp40, which can also act as a chaperone by itself (13.Langer T. Lu C. Echols H. Flanagan J. Hayer M.K. Hartl F.U. Nature. 1992; 356: 683-689Crossref PubMed Scopus (793) Google Scholar, 14.Szabo A. Langer T. Schroder H. Flanagan J. Bukau B. Hartl F.U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10345-10349Crossref PubMed Scopus (444) Google Scholar, 15.Rüdiger S. Scheneider-Mergener J. Bukau B. EMBO J. 2001; 20: 1042-1050Crossref PubMed Scopus (225) Google Scholar), consists of four conserved functional regions, as determined by genetic and mutational studies in vivo (16.Yan W. Craig E.A. Mol. Cell. Biol. 1999; 19: 7751-7758Crossref PubMed Scopus (123) Google Scholar, 17.Johnson J.L. Craig E.A. J. Cell Biol. 2001; 152: 851-856Crossref PubMed Scopus (85) Google Scholar, 18.Lee S. Fan C.Y. Younger J.M. Ren H. Cyr D.M. J. Biol. Chem. 2002; 277: 21675-21682Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 19.Fan C.Y. Lee S. Ren H.Y. Cyr D.M. Mol. Biol. Cell. 2004; 15: 761-773Crossref PubMed Scopus (92) Google Scholar) and by biophysical methods (11.Karzai A.W. MaMacken R. J. Biol. Chem. 1996; 271: 11236-11246Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 20.Szabo A. Korszun R. Hartl F.U. Flanagan J. EMBO J. 1996; 15: 408-417Crossref PubMed Scopus (276) Google Scholar, 21.Suh W-C. Lu C.Z. Gross C.A. J. Biol. Chem. 1999; 274: 30534-30539Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 22.Li J. Qian X. Sha B. Structure. 2003; 11: 1475-1483Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) (Fig. 1A). The highly conserved α-helical N-terminal domain, referred to as the J-domain, is characteristic of proteins in this family, and the binding of this domain to Hsp70 stimulates the ATPase activity of Hsp70 (11.Karzai A.W. MaMacken R. J. Biol. Chem. 1996; 271: 11236-11246Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 12.Laufen T. Mayer M.P. Beisel C. Klostermeier D. Mogk A. Reistein J. Bukau B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5452-5457Crossref PubMed Scopus (475) Google Scholar, 14.Szabo A. Langer T. Schroder H. Flanagan J. Bukau B. Hartl F.U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10345-10349Crossref PubMed Scopus (444) Google Scholar, 21.Suh W-C. Lu C.Z. Gross C.A. J. Biol. Chem. 1999; 274: 30534-30539Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 23.Greene M.K. Maskos K. Landry S.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6108-6113Crossref PubMed Scopus (248) Google Scholar). Adjacent to the J-domain is a glycine/phenylalanine-rich region (G/F-rich; Fig. 1A) that is disordered and likely to be responsible for flexibility (11.Karzai A.W. MaMacken R. J. Biol. Chem. 1996; 271: 11236-11246Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 24.Szyperski T. Pellechia M. Wall D. Georgopoulus C. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11343-11347Crossref PubMed Scopus (141) Google Scholar). The central region consists of a cysteine-rich domain (Cys_rich; Fig. 1A) that includes four repeats of the motif CXXCXGXG (where X is any amino acid) and folds in a zinc-dependent fashion with two repeats bound to one zinc ion (20.Szabo A. Korszun R. Hartl F.U. Flanagan J. EMBO J. 1996; 15: 408-417Crossref PubMed Scopus (276) Google Scholar, 25.Banecki B. Liberek K. Wall D. Wawrzynow A. Georgopoulos C. Bertoli E. Tanfani F. Zylicz M. J. Biol. Chem. 1996; 271: 14840-14848Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 26.Martinez-Yamout M. Legge G.B. Zhang O. Wright P.E. Dyson H.J. J. Mol. Biol. 2000; 300: 805-818Crossref PubMed Scopus (90) Google Scholar). The C-terminal domain (Fig. 1A) forms a β-sheet structure involved in the dimerization of Hsp40 (27.Sha B. Lee S. Cyr D.M. Structure. 2000; 8: 799-807Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). The Cys-rich and C-terminal domains are involved in substrate binding and presentation (22.Li J. Qian X. Sha B. Structure. 2003; 11: 1475-1483Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 25.Banecki B. Liberek K. Wall D. Wawrzynow A. Georgopoulos C. Bertoli E. Tanfani F. Zylicz M. J. Biol. Chem. 1996; 271: 14840-14848Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 28.Lu Z. Cyr D.M. J. Biol. Chem. 1998; 273: 5970-5978Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Hsp40 proteins occur throughout the cell and show a high diversity in eukaryotic genomes (29.Miernyk J.A. Cell Stress Chaperones. 2001; 6: 209-218Crossref PubMed Google Scholar, 30.Borges J.C. Peroto M.C. Ramos C.H.I. Genet. Mol. Biol. 2001; 24: 85-92Crossref Google Scholar), with at least 44 genes present in the human genome (31.Venter J.C. Adams M.D. Myers E.W. Li P.W. Mural R.J. Sutton G.G. Smith H.O. Yandell M. Evans C.A. Holt R.A. Science. 2001; 291: 1304-1351Crossref PubMed Scopus (10698) Google Scholar). Based on their architecture and cellular location (4.Mayer M.P. Brehmer D. Gässler C.S. Bukau B. Adv. Protein Chem. 2001; 50: 1-45Google Scholar, 32.Cyr D.M. Langer T. Douglas M.G. Trends Biochem. Sci. 1994; 19: 176-181Abstract Full Text PDF PubMed Scopus (402) Google Scholar, 33.Cheetham M.E. Caplan A.J. Cell Stress Chaper. 1998; 3: 28-36Crossref PubMed Scopus (495) Google Scholar), Hsp40 proteins are classified in three main subfamilies (A–C, also referred to as types I-III; Fig. 1A) (4.Mayer M.P. Brehmer D. Gässler C.S. Bukau B. Adv. Protein Chem. 2001; 50: 1-45Google Scholar, 33.Cheetham M.E. Caplan A.J. Cell Stress Chaper. 1998; 3: 28-36Crossref PubMed Scopus (495) Google Scholar, 34.Ohtsuka K. Hata M. Cell Stress Chaper. 2000; 5: 98-112Crossref PubMed Scopus (127) Google Scholar): subfamily A consists of proteins with the four domains described above, subfamily B contains proteins that lack the Cys-rich domain, and subfamily C has only the J-domain that is not necessarily located at the N terminus (4.Mayer M.P. Brehmer D. Gässler C.S. Bukau B. Adv. Protein Chem. 2001; 50: 1-45Google Scholar, 33.Cheetham M.E. Caplan A.J. Cell Stress Chaper. 1998; 3: 28-36Crossref PubMed Scopus (495) Google Scholar). Hsp40 proteins of subfamily A have autonomous chaperone activity and may therefore work in an Hsp70-dependent or -independent manner. In contrast, Hsp40 proteins of subfamily B have no autonomous chaperone activity and depend on Hsp70 for full activity (18.Lee S. Fan C.Y. Younger J.M. Ren H. Cyr D.M. J. Biol. Chem. 2002; 277: 21675-21682Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 33.Cheetham M.E. Caplan A.J. Cell Stress Chaper. 1998; 3: 28-36Crossref PubMed Scopus (495) Google Scholar, 35.Lu Z. Cyr D.M. J. Biol. Chem. 1998; 273: 27824-27830Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 36.Linke K. Wolfram T. Bussemer J. Jakob U. J. Biol. Chem. 2003; 278: 44457-44466Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Although high resolution structures of the C-terminal, Cys-rich, and J-domains are available, there is still little information about how these domains interact with each other, with other domains and also with Hsp70. To obtain further information about relevant structural and functional characteristics of human Hsp40 proteins, we used biophysical methods, including small angle x-ray scattering (SAXS) and analytical ultra-centrifugation (AUC), to study DjA1 (also known as Hdj2/dj2/HSDJ/Rdj1), a representative of subfamily A, its C-terminal deletion mutant (DjA1-(1–332)), and DjB4 (also known as Hlj1/DnaJW), a representative of subfamily B that shares 65% identity with DjB1/Hdj1. Cloning, Expression, and Purification—DjA1 was cloned from the human gene DNAJA1 (cDNA clone, GenBank™ accession number AW247277) and DjB4 was cloned from the human gene DNAJB4 (cDNA clone, GenBank™ accession number AA081471). The Hsp40 nomenclatures DjA1 and DjB4 were used as described by Ohtsuka and Hata (34.Ohtsuka K. Hata M. Cell Stress Chaper. 2000; 5: 98-112Crossref PubMed Scopus (127) Google Scholar). Two primers were used to amplify the DjA1 cDNA by PCR and to create restriction enzyme sites for NdeI and XhoI: a DjA1 5′-primer (5′-CCGGCAGGCTAGCATGGTGAAAGAAACAAC-3′) containing an NdeI restriction site and a DjA1 3′-primer (5′-TGAGTGTTATTCTCGAGTCATTAAGAGGTCTG-3′) containing an XhoI restriction site. Two primers were used to amplify the DjB4 cDNA by PCR and to create restriction enzyme sites for NdeI and BamHI: a DjB4 5′-primer (5′-TCAAGGCATTCCATATGGGGAAAGACTATTA-3′) containing an NdeI restriction site and a DjB4 3′-primer (5′-GTGTAACAAAGTGGATCCTACTATGAGGCAGG-3′) containing a BamHI restriction site. The C-terminal deletion of DjA1 was constructed by site-directed mutagenesis using a primer that created a stop codon at residue Phe333 followed by a restriction site for XhoI (5′-TTATCAGGCTCGAGTTAGCCATTCTC-3′). The PCR products were cloned into the pET28A expression vector (Novagen) for His tag purification methods. The correct cloning was confirmed by DNA sequencing using an ABI 377 Prism system (PerkinElmer Life Sciences). These procedures created the vectors pET28aDjA1, pET28aDjB4, and pET28aDjA1-(1–332), which were transformed into the Escherichia coli strain BL21(DE3) for heterologous protein expression by adding isopropyl thio-β-d-galactoside (0.4 mm) at A600 = 0.6. The induced cells were grown for 5 h and harvested by centrifugation for 10 min at 2,600 × g. The bacterial pellet was resuspended in lysis buffer (50 mm Tris-HCl, pH 8.0, 500 mm KCl and 10 mm EDTA; 15 ml of buffer/liter of medium) in the presence of 5 units of DNase (Invitrogen, Life Technologies, Inc.) and 30 μg of lysozyme/ml (Sigma) for 30 min. The pellet was then disrupted by sonication in an ice bath, and centrifuged as described above. The supernatant was fractionated by metal affinity chromatography in a HiTrap chelating column (Amersham Biosciences) using an AKTA FPLC system (Amersham Biosciences). The proteins were eluted with imidazole (500 mm) and loaded onto a HiLoad Superdex 200-pg column (2.6 cm × 60 cm; Amersham Biosciences) using an AKTA FPLC system. The degree of purification was estimated by SDS-PAGE, and the protein concentration was determined spectrophotometrically using a calculated extinction coefficient for denatured proteins (37.Edelhock H. Biochemistry. 1967; 6: 1948-1954Crossref PubMed Scopus (3007) Google Scholar, 38.Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5073) Google Scholar). Analytical Molecular Exclusion Chromatography—Analytical molecular exclusion chromatography was done using a Superose 12 HR 10/30 (Amersham Biosciences) column coupled to an AKTA Purifier system (Amersham Biosciences). The column was equilibrated with 25 mm Tris-HCl, pH 7.5, containing 500 mm NaCl, 1% glycerol, and β-mercaptoethanol (1–10 mm). The column was washed with two column volumes of this same buffer at a flow rate of 0.5 ml/min, and aliquots of proteins in 100 μl were loaded onto the column. The elution profile was determined by monitoring the absorbance at 280 nm. The apparent molecular mass was calculated using a plot of ln of the molecular mass (kDa) of standard proteins (thyroglobulin, 669 kDa; γ-globulin, 160 kDa; bovine serum albumin, 69 kDa; chicken ovalbumin, 45 kDa; cytochrome c, 12 kDa) versus the retention time. Circular Dichroism Spectroscopy—Circular dichroism (CD) measurements were done using a Jasco J-810 spectropolarimeter with the temperature controlled by a Peltier-type control system PFD 425S. Hsp40 proteins were resuspended in 25 mm Tris-HCl, pH 7.5, containing 500 mm NaCl and 1 mm β-mercaptoethanol at final concentrations of 20–50 μm. The data were collected at a scanning rate of 50 nm/min with a spectral bandwidth of 1 nm using a 0.1-mm path length cell. CDNN deconvolution software (Version 2, Bioinformatik.biochemtech. uni-halle.dee/cdnn) was used for secondary structure prediction. All buffers used were of analytical grade and were filtered before use to avoid light scattering by small particles. Measurement of Chaperone Activity—The method described by Lu and Cyr (28.Lu Z. Cyr D.M. J. Biol. Chem. 1998; 273: 5970-5978Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 35.Lu Z. Cyr D.M. J. Biol. Chem. 1998; 273: 27824-27830Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) was used to assess the ability of Hsp40 proteins to interact with unfolded proteins. Briefly, luciferase (Promega) was chemically denatured by diluting in guanidinium-HCl (6 m) for 40 min at room temperature and then diluted 25× in 50 μl of 25 mm Tris-HCl, pH 7.5, containing 500 mm NaCl and 5 mm β-mercaptoethanol in the absence and presence of His-tagged Hsp40 proteins (see figure captions for details of the concentrations). In another protocol, luciferase (2 μm) was thermally denatured by incubation at 42 °C for 10 min in the absence and presence of His-tagged Hsp40 proteins (see figure captions for details of the concentrations) and then cooled to room temperature. The solutions were centrifuged for 10 min at 21,000 × g, and 50 μlofthe supernatant was incubated with 50 μl of a 50% slurry of Talon metal chelate resin (Clontech) in 25 mm Tris-HCl, pH 7.5, containing 500 mm NaCl and 5 mm β-mercaptoethanol for 1 h at room temperature. The mixture was then centrifuged for 1 min at 10,000 × g and 4 °C, and the pellet containing the resin was washed twice with 75 μl of the Tris buffer indicated above containing 15 mm imidazole. The protein complexes bound to the resin were eluted with this same Tris buffer containing 150 mm imidazole and then concentrated by precipitation with acetone 80% (Merck) and visualized by SDS-PAGE. Analytical Ultracentrifugation—The sedimentation velocity and sedimentation equilibrium experiments were done with a Beckman Optima XL-A analytical ultracentrifuge. The proteins DjA1, DjA1-(1–332), and DjB4 were tested at concentrations from 50 to 1000 μg/ml in 25 mm Tris-HCl, pH 7.5, containing 500 mm NaCl, 1% glycerol (but not for DjA1-(1–332)) and 1 mm β-mercaptoethanol, with no apparent aggregation. The sedimentation velocity experiments were done at 20 °C, 25,000 rpm for DjA1 and DjB4, and 30,000 and 40,000 rpm for DjA1-(1–332) (AN-60Ti rotor), and the scan data were acquired at 230 and 238 nm for low and high protein concentrations, respectively. The sedimentation equilibrium experiments were done at 20 °C at speeds of 6,000, 8,000, and 10,000 rpm with the AN-60Ti rotor and scan data acquisition at 238 nm. Analysis of the data involved the fitting of a model of absorbance versus cell radius data using nonlinear regression. All fittings were done using the Origin software package (Microcal Software) supplied with the instrument. The van Holde-Weischet (39.Van Holde K.E. Weischet W.O. Biopolymers. 1978; 17: 1397-1403Crossref Scopus (318) Google Scholar) (sediment coefficient plot) and the sedimentation time derivative (g(s*) integral distribution) (40.Stafford W.F. Methods Enzymol. 1994; 240: 478-501Crossref PubMed Scopus (121) Google Scholar) methods were used to analyze the sedimentation velocity experiments. The methods used to analyze the velocity and equilibrium experiments allowed the calculation of the apparent sedimentation coefficient s, the diffusion coefficient D, and the molecular mass M. The ratio of the sedimentation to diffusion coefficients gave the molecular mass in Equation 1, M=sRTD(1−Vbarρ) (Eq. 1) where R is the gas constant and T is the absolute temperature. The Sednterp software (www.jphilo.mailway.com/download.htm) was used to estimate the partial specific volume of the proteins at 20 °C (VbarDjA1 = 0.7275 ml/g, VbarDjA1-(1–332) = 0.7309 ml/g, and VbarDjB4 = 0.7302 ml/g), the buffer density (ρ = 1.02163 g/ml) and the buffer viscosity (η = 1.0851 × 10–2 poise). The self-association method was used to analyze the sedimentation equilibrium experiments, and several models of association for DjA1 and DjB4 were used to fit the data. The distribution of each protein throughout the cell, as determined in the equilibrium sedimentation experiments, was fitted with Equation 2 (41.Johnson M.L. Correia J.J. Yphantis D.A. Halvorson H.R. Biophys. J. 1981; 36: 575-588Abstract Full Text PDF PubMed Scopus (778) Google Scholar), C=C0exp[M(1−Vbarρ)ω2(r2−r0)2RT] (Eq. 2) where C is the protein concentration at radial position r, C0 is the protein concentration at radial position r0, and ω is the centrifugal angular velocity. The Sednterp software was used to estimate the standard sedimentation coefficients (s20,w) at each protein concentration and to calculate s20,w0 by extrapolation to a protein concentration of 0 mg/ml. This procedure corrects for effects of temperature, solution viscosity, and molecular crowding (42.Laue T.M. Curr. Opin. Struct. Biol. 2001; 11: 579-583Crossref PubMed Scopus (46) Google Scholar). SAXS—The SAXS experiments were done at the SAS beamline of the LNLS synchrotron radiation facility in Campinas, Brazil (43.Kellermann G. Vicentin F. Tamura E. Rocha M. Tolentino H. Barbosa A. Craievich A. Torriani I. J. Appl. Crystallogr. 1997; 30: 880-883Crossref Google Scholar) under the same conditions as those described by Borges et al. (44.Borges J.C. Fischer H. Craievich A.F. Hansen L.D. Ramos C.H.I. J. Biol. Chem. 2003; 278: 35337-35344Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The measurements were done using a monochromatic x-ray beam with a wavelength λ = 1.488 Å. For a sample-to-detector distance of 840 mm selected for the experiments, the modulus of the photon momentum transfer (q = 4πsinθ/λ, 2θ being the scattering angle) covered a range from q = 0.01 Å–1 to q = 0.44 Å–1. The scattering intensity curves, I(q), were recorded using a gas 1 dimensional position sensitive x-ray detector. To monitor possible effects from radiation damage and synchrotron beam instabilities, the SAXS curves were determined in many short frames (90 s each). The SAXS data obtained were normalized to account for the natural decay in intensity of the synchrotron incident beam and were corrected for non-homogeneous detector responses. Finally, the scattering intensity produced by the buffer was subtracted, and the difference curves were scaled to give equivalent protein concentrations. Three types of samples with different concentrations were studied by SAXS: 1) 3.3 mg/ml DjA1 in 25 mm Tris-HCl, pH 7.5, containing 500 mm NaCl, 1% glycerol and 1 mm dithiothreitol, 2) 2.9 and 6.5 mg/ml of DjA1-(1–332) in 25 mm Tris-HCl, pH 7.5, containing 500 mm NaCl and 1 mm β-mercaptoethanol, and 3) 2.5, 4.4, 6.2, and 8.3 mg/ml DjB4 in 25 mm Tris-HCl, pH 7.5, containing 500 mm NaCl and 1 mm β-mercaptoethanol. Reliable structural information about the low resolution structure of proteins in solution can be derived from SAXS data provided that all proteins are in the same (monomeric or oligomeric) state and that the solution is “dilute,” i.e. interference effects in scattering amplitudes produced by different proteins are negligible. Under these assumptions, the total scattering amplitude is proportional to the form factor of an isolated (monomeric or oligomeric) protein averaged for all orientations. When samples with a high concentration were available, data merging was done using low and high concentration samples for the high and low q ranges of the scattering intensity, respectively. This procedure reduced the overall statistical error in the scattering curves without introducing unwanted interferences or spatial correlation effects in the small q range. To establish the molecular masses of DjA1, DjA1-(1–332), and DjB4, the SAXS intensity produced by a solution of bovine serum albumin (69 kDa, 5 and 10 mg/ml) was also determined. Since, for dilute solutions, the normalized intensity extrapolated to q = 0, I(0), is proportional to the molecular mass, the molecular masses (M) of the proteins investigated here were determined from the ratios between the I(0) values corresponding to the different samples and that of the standard bovine serum albumin. Computer Programs—By applying the indirect Fourier transform program GNOM (45.Svergun D.I. J. Appl. Crystallogr. 1992; 25: 495-503Crossref Scopus (2987) Google Scholar) to the normalized SAXS curves, the distance distribution function p(r) and the radius of gyration, Rg, of the proteins studied were evaluated (46.Porod G. Glatter O. Kratky O. Small Angle X-ray Scattering. Academic Press, London1982: 17-51Google Scholar). Prior to GNOM analysis, a constant intensity background was subtracted from the SAXS data to ensure that the intensity at higher angles decayed as q–4, according to Porod's law for a two-electron density model (46.Porod G. Glatter O. Kratky O. Small Angle X-ray Scattering. Academic Press, London1982: 17-51Google Scholar). The low resolution shapes (or molecular envelopes) of the proteins were restored from the experimental SAXS curves using an ab initio method named DAMMIN (47.Svergun D.I. Biophys. J. 1999; 76: 2879-2886Abstract Full Text Full Text PDF PubMed Scopus (1756) Google Scholar) and recently described in Borges et al. (44.Borges J.C. Fischer H. Craievich A.F. Hansen L.D. Ramos C.H.I. J. Biol. Chem. 2003; 278: 35337-35344Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Briefly, in this method, the low resolution shape of the protein was simulated by a set of small spheres that initially filled another sphere with a diameter equal to the maximum diameter, Dmax, of the protein being studied (previously determined using GNOM). DAMMIN yielded a structural model containing a fraction of the initial number of dummy atoms whose associated scattering intensity gave the best fit to the SAXS data. The configuration of the different protein domains could be refined by manually rotating or displacing the domains until the discrepancy between the calculated and experimental SAXS curves was minimized. The protein models were displayed using the program WebLab ViewerLite software (www.accelrys.com). The HydroPro software (48.Garcia de la Torre J. Huertas M.L. Carrasco B. Biophys. J. 2000; 78: 719-730Abstract Full Text Full Text PDF PubMed Scopus (891) Google Scholar) was used to estimate the translational diffusion coefficient Dt, the radii of gyration Rg, the sedimentation coefficient s, and the maximum distance (Dmax) starting from the ab initio models generated by DAMMIN using SAXS data obtained at 20 °C. The HydroPro software was configured with the radius of the atomic elements from the ab initio analysis, with sigma factors from 5 to 8 (as indicated by the supplier) and a minibeads radius from 6 to 2 Å (SIGMIN and SIGMAX), after an initial evaluation of the two extremes. The parameters Vbar, ρ, and η were estimated using the software Sednterp as described above. The translational fraction ratio or Perrin factor P, which indicates the relationship between the frictional coefficient of the Hsp40 particles and a sphere of the same molecular mass (f/f0), was estimated using Solpro software (49.Garcia de la Torre J. Carrasco B. Harding S.E. Eur. Biophys. J. 1997; 25: 361-372Crossref PubMed Scopus (65) Google Scholar). Purification of DjA1 and DjB4 as Folded Dimers and of DjA1-(1–332) as a Folded Monomer—The correct cloning and sequencing of DjA1, DjB4, and DjA1-(1–332) were confirmed by DNA sequencing. All of the proteins were expressed in large quantities and were more than 95% pure as confirmed by SDS-PAGE (Fig. 2A). The proteins were unstable at low ionic strength but were soluble in the presence of 500 mm NaCl. DjA1 and DjB4 were purified as dimers and the mutant DjA1-(1–332) was purified as a monomer, as confirmed by analytical molecular exclusion chromatography (Fig. 2B). The folding state of the proteins was investigated by CD, and the resulting spectra corresponded to folded proteins with a minimum at 208 nm (Fig. 2C). The analysis using the CDNN deconvolution software indicated that the proteins had similar amounts of secondary structure: 35% β-sheet structure and 10% α-helices. The high content of β-sheet structure agreed with x-ray crystallography and nuclear magnetic resonance data indicating the presence of β-sheet structures in the C terminus (22.Li J. Qian X. Sha B. Structure. 2003; 11: 1475-1483Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 27.Sha B. Lee S. Cyr D.M. Structure. 2000; 8: 799-807Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scho" @default.
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• W2090161921 date "2005-04-01" @default.
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• W2090161921 title "Low Resolution Structural Study of Two Human HSP40 Chaperones in Solution" @default.
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• W2090161921 doi "https://doi.org/10.1074/jbc.m408349200" @default.
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