Produção Nacional
Revisado por pares
Free Human Mitochondrial GrpE Is a Symmetric Dimer in Solution
2003; Elsevier BV; Volume: 278; Issue: 37 Linguagem: Inglês
10.1074/jbc.m305083200
ISSN1083-351X
AutoresJúlio C. Borges, Hannes Fischer, A. F. Craievich, Lee D. Hansen, Carlos H.I. Ramos,
Tópico(s)ATP Synthase and ATPases Research
ResumoThe co-chaperone GrpE is essential for the activities of the Hsp70 system, which assists protein folding. GrpE is present in several organisms, and characterization of homologous GrpEs is important for developing structure-function relationships. Cloning, producing, and conformational studies of the recombinant human mitochondrial GrpE are reported here. Circular dichroism measurements demonstrate that the purified protein is folded. Thermal unfolding of human GrpE measured both by circular dichroism and differential scanning calorimetry differs from that of prokaryotic GrpE. Analytical ultracentrifugation data indicate that human GrpE is a dimer, and the sedimentation coefficient agrees with an elongated shape model. Small angle x-ray scattering analysis shows that the protein possesses an elongated shape in solution and demonstrates that its envelope, determined by an ab initio method, is similar to the high resolution envelope of Escherichia coli GrpE bound to DnaK obtained from single crystal x-ray diffraction. However, in these conditions, the E. coli GrpE dimer is asymmetric because the monomer that binds DnaK adopts an open conformation. It is of considerable importance for structural GrpE research to answer the question of whether the GrpE dimer is only asymmetric while bound to DnaK or also as a free dimer in solution. The low resolution structure of human GrpE presented here suggests that GrpE is a symmetric dimer when not bound to DnaK. This information is important for understanding the conformational changes GrpE undergoes on binding to DnaK. The co-chaperone GrpE is essential for the activities of the Hsp70 system, which assists protein folding. GrpE is present in several organisms, and characterization of homologous GrpEs is important for developing structure-function relationships. Cloning, producing, and conformational studies of the recombinant human mitochondrial GrpE are reported here. Circular dichroism measurements demonstrate that the purified protein is folded. Thermal unfolding of human GrpE measured both by circular dichroism and differential scanning calorimetry differs from that of prokaryotic GrpE. Analytical ultracentrifugation data indicate that human GrpE is a dimer, and the sedimentation coefficient agrees with an elongated shape model. Small angle x-ray scattering analysis shows that the protein possesses an elongated shape in solution and demonstrates that its envelope, determined by an ab initio method, is similar to the high resolution envelope of Escherichia coli GrpE bound to DnaK obtained from single crystal x-ray diffraction. However, in these conditions, the E. coli GrpE dimer is asymmetric because the monomer that binds DnaK adopts an open conformation. It is of considerable importance for structural GrpE research to answer the question of whether the GrpE dimer is only asymmetric while bound to DnaK or also as a free dimer in solution. The low resolution structure of human GrpE presented here suggests that GrpE is a symmetric dimer when not bound to DnaK. This information is important for understanding the conformational changes GrpE undergoes on binding to DnaK. Nascent proteins in the cell sometimes require the assistance of one or more protein complexes named molecular chaperones to fold correctly (1Martin J. Hartl F.U. Curr. Opin. Struct. Biol. 1997; 7: 41-45Crossref PubMed Scopus (165) Google Scholar, 2Fink A.L. Physiol. Rev. 1999; 79: 425-449Crossref PubMed Scopus (873) Google Scholar). An important chaperone complex is composed of the molecular chaperones Hsp70, 1The abbreviations used are: Hsp, heat shock protein; D max, maximum diameter; DAM, dummy atom model; DSC, differential scanning calorimetry; DTT, dithiothreitol; NBD, nucleotide binding domain of Hsp70; R g, radius of gyration; SAXS, small angle x-ray scattering analysis; SBD, substrate binding domain of Hsp70. Hsp40 (or DnaK and DnaJ, respectively) and GrpE, which are highly expressed and important for several cell processes (3Friedman D.I. Olson E.R. Georgopoulos C. Tilly K. Herskowitz I. Banuett F. Microbiol. Rev. 1984; 48: 299-325Crossref PubMed Google Scholar, 4Skowyra D. Georgopoulos C. Zylicz M. Cell. 1990; 62: 939-944Abstract Full Text PDF PubMed Scopus (328) Google Scholar, 5Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3607) Google Scholar, 6Schroder H. Langer T. Hartl F.U. Bukau B. EMBO J. 1993; 12: 4137-4144Crossref PubMed Scopus (501) Google Scholar, 7Mayer M.P. Brehmer D. Gässler C.S. Bukau B. Adv. Protein Chem. 2001; 59: 1-44Crossref PubMed Scopus (134) Google Scholar). The Hsp70 affinity for unfolded proteins is regulated by nucleotide binding to its nucleotide binding domain (NBD), which has a molecular mass of about 45 kDa (5Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3607) Google Scholar, 8Flaherty K.M. DeLuca-Flaherty C. McKay D.B. Nature. 1990; 346: 623-628Crossref PubMed Scopus (833) Google Scholar). The Hsp70 C terminus forms the substrate binding domain (SBD), which is capable of binding hydrophobic amino acid residues and has a molecular mass of about 20 kDa (8Flaherty K.M. DeLuca-Flaherty C. McKay D.B. Nature. 1990; 346: 623-628Crossref PubMed Scopus (833) Google Scholar, 9Pelham H.R. Cell. 1986; 46: 959-961Abstract Full Text PDF PubMed Scopus (1147) Google Scholar). Co-chaperones Hsp40 and GrpE interact both in vivo and in vitro with DnaK (6Schroder H. Langer T. Hartl F.U. Bukau B. EMBO J. 1993; 12: 4137-4144Crossref PubMed Scopus (501) Google Scholar, 10Johnson C. Chandrasekhar G.N. Georgopoulos C. J. Bacteriol. 1989; 171: 1590-1596Crossref PubMed Google Scholar, 11Liberek K. Marszalek J. Ang D. Georgopoulos C. Zylicz M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2874-2878Crossref PubMed Scopus (693) Google Scholar), stimulating its ATPase activity (12Szabo 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) and regulating the ability of DnaK to bind and stabilize unfolded proteins (13Ang D. Chandrasekhar G.N. Zylicz M. Georgopoulos C. J. Bacteriol. 1986; 167: 25-29Crossref PubMed Google Scholar). The importance of GrpE in the Hsp70 chaperone machinery is shown by the following: it is essential for bacterial viability at all temperatures (14Langer T. Lu C. Echols H. Flanagan J. Hayer M.K. Hartl F.U. Nature. 1992; 356: 683-689Crossref PubMed Scopus (793) Google Scholar), GrpE acts as an exchange factor that releases nucleotides bound to DnaK (12Szabo 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), and it is important for DnaK recycling (11Liberek K. Marszalek J. Ang D. Georgopoulos C. Zylicz M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2874-2878Crossref PubMed Scopus (693) Google Scholar). The first indication that GrpE was a homodimer came from cross-linking studies with glutaraldehyde (15Osipiuk J. Georgopoulos C. Zylicz M. J. Biol. Chem. 1993; 268: 4821-4827Abstract Full Text PDF PubMed Google Scholar). Subsequently, analytical ultracentrifugation experiments (16Schönfeld H.J. Schmidt D. Schröder H. Bukau B. J. Biol. Chem. 1995; 270: 2183-2189Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) showed that GrpE has a dimeric structure with an elongated shape that binds DnaK with 2:1 stoichiometry. The crystallographic high resolution structure of residues 34–197 of Escherichia coli GrpE (EcGrpE34–197) complexed with the E. coli DnaK-NBD (EcDnaK3–383) corroborates that GrpE forms a dimer and shows that only one of the subunits, known as the proximal monomer, binds to Hsp70 (17Harrison C.J. Hayer-Hartl M. Di Liberto M. Hartl F.U. Kuriyan J. Science. 1997; 276: 431-435Crossref PubMed Scopus (413) Google Scholar). In this structure, the GrpE dimer is asymmetric because the proximal monomer adopts a more open conformation than the distal monomer. Knowing whether this asymmetric conformation remains while GrpE is free in solution is necessary for understanding this protein structure-function relationship in the cell. The GrpE C-terminal domain, EcGrpE141–197, is β-structured and binds to the DnaK-NBD causing the release of ADP from the NBD (17Harrison C.J. Hayer-Hartl M. Di Liberto M. Hartl F.U. Kuriyan J. Science. 1997; 276: 431-435Crossref PubMed Scopus (413) Google Scholar). The function of the GrpE N terminus remains to be understood fully. The GrpE40–86 domain forms a long coil-coiled α-helical structure (17Harrison C.J. Hayer-Hartl M. Di Liberto M. Hartl F.U. Kuriyan J. Science. 1997; 276: 431-435Crossref PubMed Scopus (413) Google Scholar) and may function as a thermosensor because it appears to be responsible for the GrpE thermal transition at physiological temperatures (18Gelinas A.D. Langsetmo K. Toth J. Bethoney K.A. Stafford W.F. Harrison C.J. J. Mol. Biol. 2002; 323: 131-142Crossref PubMed Scopus (35) Google Scholar). GrpE appears to be the only component of the Hsp70 chaperone machinery which undergoes a thermal transition at a physiologically relevant temperature (19Grimshaw J.P. Jelesarov I. Schönfeld H.J. Christen P. J. Biol. Chem. 2001; 276: 6098-6104Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The GrpE89–137 domain forms a four-helix bundle (17Harrison C.J. Hayer-Hartl M. Di Liberto M. Hartl F.U. Kuriyan J. Science. 1997; 276: 431-435Crossref PubMed Scopus (413) Google Scholar) and likely acts as the stabilization center for dimerization (18Gelinas A.D. Langsetmo K. Toth J. Bethoney K.A. Stafford W.F. Harrison C.J. J. Mol. Biol. 2002; 323: 131-142Crossref PubMed Scopus (35) Google Scholar). GrpE is present in eukaryotes (20Naylor D.J. Stines A.P. Hoogenraad N.J. Hoj P.B. J. Biol. Chem. 1998; 273: 21169-21177Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 21Schlicher T. Soll J. Plant Mol. Biol. 1997; 33: 181-185Crossref PubMed Scopus (32) Google Scholar, 22Borges J.C. Peroto M.C. Ramos C.H.I. Gen. Mol. Biol. 2001; 24: 85-92Crossref Google Scholar), and it is generally assumed that they share high structural similarity with their prokaryote homologs. Thus, it is important to characterize the structure and function of GrpE from diverse organisms to test this hypothesis. The sequence of human mitochondrial GrpE (23Choglay A.A. Chapple J.P. Blatch G.L. Cheetham M.E. Gene (Amst.). 2001; 267: 125-134Crossref PubMed Scopus (17) Google Scholar) is represented in Fig. 1 along with the E. coli GrpE sequence showing that they share about 30% identity. The mitochondrial form of human GrpE is expressed as an immature protein that matures when its mitochondrial signaling peptide is lost. Human GrpE was cloned and expressed in E. coli and purified by ion exchange chromatography and preparative molecular exchange chromatography. The secondary structure characterized by circular dichroism (CD) showed that the protein is folded. Temperature-induced unfolding followed either by CD or by differential scanning calorimetry (DSC) showed that human GrpE unfolding is only partially reversible, whereas E. coli GrpE exhibits reversible unfolding up to 60 °C (19Grimshaw J.P. Jelesarov I. Schönfeld H.J. Christen P. J. Biol. Chem. 2001; 276: 6098-6104Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Analytical ultracentrifugation data indicate that the protein has the molecular mass of a dimer, and the calculated sedimentation coefficient agrees with the value expected for a protein with an elongated shape. The elongated shape was also derived independently from small angle x-ray scattering analysis (SAXS). The hydrodynamic parameters derived from SAXS data agree with those determined by analytical ultracentrifugation. Our envelope models are similar to the envelope of the crystallographic structure of E. coli GrpE bound to the DnaKNBD determined by Harrison et al. (17Harrison C.J. Hayer-Hartl M. Di Liberto M. Hartl F.U. Kuriyan J. Science. 1997; 276: 431-435Crossref PubMed Scopus (413) Google Scholar). The low resolution structure generated from our SAXS data in solution suggests that GrpE is a symmetric dimer when not bound to DnaK. The implications of our conclusions concerning human GrpE conformational changes in structure-function relationships of this co-chaperone are discussed. Cloning, Expression, and Purification—The human cDNA IMAGE clone (GenBank accession number BE614754) was used for cloning the mitochondrial GrpE (Mt-GrpE#1, GenBank accession number Q9HAV7). Two primers were used to amplify the cDNA by PCR and to create the restriction enzyme sites NdeI and XhoI for cloning into pET23a vector (Novagen). The 5′-primer (5′-TCTCCCCGGCATATGTGCACAG-3′) containing the NdeI restriction site was designed to anneal downstream to the mitochondrial peptide signal, eliminating this sequence in the recombinant protein. The correct cloning was confirmed by DNA sequencing using an ABI 377 Prism system (PerkinElmer Life Sciences). These procedures created the pET23aHMGrpE#1 vector, which was transformed in E. coli strain BL21(DE3) for protein expression by adding 0.4 mmol/liter isopropyl thio-β-d-galactoside at A 600 = 0.8. 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 mmol/liter Tris-HCl, pH 8.0, 50 mmol/liter KCl, 10 mmol/liter EDTA, 15 ml/liter medium), disrupted by sonication in an ice bath, and centrifuged as described above. The supernatant was dialyzed against equilibration buffer (25 mmol/liter Tris-HCl, pH 7.5, 1 mmol/liter DTT) and then submitted to ion exchange chromatography on Q-Sepharose resin using an ÄKTA FPLC (Amersham Biosciences). Human GrpE was eluted in 100 mmol/liter NaCl, dialyzed against the second equilibration buffer (25 mmol/liter Tris-HCl, pH 7.5, 150 mmol/liter NaCl, 1 mmol/liter DTT) and loaded on a HiLoad Superdex 200pg molecular exclusion column using an ÄKTA FPLC. The degree of purification of human GrpE was estimated by SDS-PAGE (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar), and its concentration was determined spectrophotometrically using the calculated extinction coefficient for denatured proteins (25Edelhock H. Biochemistry. 1967; 6: 1948-1954Crossref PubMed Scopus (3007) Google Scholar, 26Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5073) Google Scholar). Circular Dichroism (CD) Spectroscopy—CD measurements were recorded on a Jasco J-810 spectropolarimeter with temperature controlled by Peltier Type Control System PFD 425S. Human GrpE was resuspended in 10 mmol/liter phosphate buffer, pH 7.2, and 1 mmol/liter β-mercaptoethanol at a final concentration of 20 μmol/liter. Data were collected at a scanning rate of 50 nm/min with spectral band width of 1 nm using a 1-mm path length cell at increasing temperatures (for details, see Fig. 3 legend). CDNN Deconvolution software (version 2; Bioinformatik.biochemtech.uni-halle.dee/cdnn) was employed for secondary structure prediction. All buffers used were of chemical grade and were filtered before use to avoid scattering from small particles. DSC—The measurements of human GrpE thermal denaturation were done both in an N-DSC III differential scanning calorimeter (Calorimetry Sciences Corp.) and in a VP-DSC Differential Scanning Calorimeter (MicroCal), which gave similar results. The measurements were performed with protein concentrations of 7, 45, and 130 μmol/liter and in two sets of buffers; 25 mmol/liter Hepes pH 7.6, 50 mmol/liter KCl, 5 mmol/liter MgCl2; and 25 mmol/liter Tris-HCl, 150–500 mmol/liter NaCl, 1 mmol/liter β-mercaptoethanol. The scan rate was varied from 0.5 to 1.5 °C/min, and the temperature measurement range was from 10 to 100 °C. The reversibility of unfolding was tested by performing several consecutive up and down scans and by scan rate variation. Base lines were run several times in all of the conditions mentioned above. Analytical Ultracentrifugation—Sedimentation velocity and sedimentation equilibrium experiments were performed with a Beckman Optima XL-A analytical ultracentrifuge. The protein was tested in concentrations of 50, 100, and 200 μg/ml in 25 mmol/liter Tris-HCl buffer at pH 7.5, with 150 mmol/liter NaCl and 0.5 mmol/liter DTT, with no apparent aggregation. The sedimentation velocity experiments were carried out at 20 °C, 40,000 rpm (AN-60Ti rotor), and the scan data acquisition was taken at 230 nm. The sedimentation equilibrium experiments were made at 20 °C at speeds of 7,000, 9,000, and 11,000 rpm using the AN-60Ti rotor. Scan data acquisition was done at 230 nm. The analysis involved fitting a model of absorbance versus cell radius data by nonlinear regression. All fits were done with the ORIGIN software package (MicroCal Software) supplied with the instrument. The van Holde-Weischet (27van Holde K.E. Weischet W.O. Biopolymers. 1978; 17: 1397-1403Crossref Scopus (318) Google Scholar) (sediment coefficient plot), Second Moment (28Goldberg R.J. J. Phys. Chem. 1953; 57: 194-202Crossref Scopus (151) Google Scholar), and the Sedimentation Time Derivative (g(s*) integral distribution) (29Stafford 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 for analyzing both velocity and equilibrium experiments allow the calculation of the apparent sedimentation coefficient s, the diffusion coefficient D, and the molecular weight M. The ratio of the sedimentation to diffusion coefficient gives the molecular weight; M=sRTD(1-Vbarρ)(Eq. 1) R is the gas constant and T is the absolute temperature. The software Sednterp (www.jphilo.mailway.com/download.htm) was used to estimate protein partial specific volume (Vbar = 0.7432 ml/g), buffer density (ρ = 1.0052 g/ml), and buffer viscosity (η = 0.01poise). The Self-association method was used to analyze the sedimentation equilibrium experiments using several models of association for human GrpE to fit the data. The distribution of the protein along the cell, obtained in the equilibrium sedimentation experiments, was fitted with the Equation 2 (30Johnson 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=C0expM(1-Vbarρ)ω2(r2-r0)2RT(Eq. 2) where C is the protein concentration at radial position r, C 0 is the protein concentration at radial position r 0, and ω is the centrifugal angular velocity. SAXS—Small angle x-ray scattering experiments were performed at the SAS beam line of the LNLS synchrotron radiation facility, Campinas, Brazil (31Kellermann G. Vicentin F. Tamura E. Rocha M. Tolentino H. Barbosa A. Craievich A. Torriani I. J. Appl. Crystallogr. 1997; 30: 880-883Crossref Google Scholar). Measurements were make with a monochromatic x-ray beam with a wavelength λ = 1.488 Å for a sample-detector distance of 840 mm covering the momentum transfer range 0.01 < q < 0.44 Å-1 (q = 4πsinθ/λ, where 2θ is the scattering angle). The scattering intensity was recorded using a one-dimensional position-sensitive x-ray detector. The scattering curves produced by the protein solutions of either 5.0 or 19.4 mg/ml and of the solvent (25 mm Tris-HCl, pH 7.5, and 1 mmol/liter DTT) were collected in many short (90 s) frames to monitor radiation damage and beam stability. The data were normalized to account for the natural decay in intensity of the synchrotron incident beam and corrected for inhomogeneous detector response. The scattering intensity produced by the buffer was subtracted, and the difference curves were scaled to equivalent protein concentration. To determine the molecular mass of human GrpE, a 5 mg/ml bovine serum albumin (66 kDa) solution was used as a standard. The molecular mass of human GrpE was inferred from the ratio of the extrapolated I(0) value of human GrpE to that of bovine serum albumin. Because there was no negative region in the pair distribution function, we can safely conclude that all solutions studied were in the "dilute" state, i.e. no interferences of scattering amplitudes were produced by the interaction of different isolated scattering objects. Models and Computer Programs—The distance distribution function p(r) and the radius of gyration R g of the human GrpE protein were evaluated from the corrected and normalized SAXS curves by the indirect Fourier transform program GNOM (32Svergun D.I. J. Appl. Crystallogr. 1992; 25: 495-503Crossref Scopus (2987) Google Scholar, 33Svergun D.I. Stuhrmann H.B. Acta Crystallogr. Sect. A. 1991; 47: 736-744Crossref Scopus (128) Google Scholar). A constant was subtracted from the experimental data to ensure that the intensity at higher angles decayed as q –4 following Porod's law for a two-electron density model (34Porod G. Glatter O. Kratky O. Small-angle X-ray Scattering. Academic Press, London1982: 17-51Google Scholar). The value of the constant was derived automatically from the outer part of the curve using a linear fit to q 4I(q) versus q4 plots by the shape determination program DAMMIN (35Svergun D.I. Biophys. J. 1999; 76: 2879-2886Abstract Full Text Full Text PDF PubMed Scopus (1756) Google Scholar), which will be described later. This procedure reduces the contribution from scattering because of the short range fluctuations of the internal protein structure and yields an approximation of the "shape scattering curve" (i.e. the scattering intensity produced by the excluded volume of a particle with a spatially constant density). The low resolution shape of GrpE was obtained from the experimental SAXS data with an ab initio method implemented in DAMMIN (34Porod G. Glatter O. Kratky O. Small-angle X-ray Scattering. Academic Press, London1982: 17-51Google Scholar). A sphere of diameter D max was filled by a regular grid of points corresponding to a dense hexagonal packing of small spheres (dummy atoms) of radius r 0 ≪ D max. The structure of the dummy atom model (DAM) is defined by a configuration X, assigning an index to each atom corresponding to solvent {0} or solute particle {1}. The computed scattering intensity curve of the DAM is compared with the intensity curve determined experimentally and the model is progressively modified by successive minimization trials of a function f(X) defined as follows, f(X)=χ2+αP(X)(Eq. 3) where χ is the discrepancy between the experimental and modeled SAXS intensity functions given by the following, χ=1N-1∑j=1NI(qj)-Iexp(qj)σ(qj)2(Eq. 4) where N is the number of experimental points, I exp(qj) is the experimental intensity, and σ(qj) is the standard deviation in the jth point. In Equation 3, α is a positive constant, and P(X) is an added penalty function that avoids solutions with loose bounds or disconnected structures. The DAMMIN program searches for a compact interconnected configuration χ, minimizing the function f(X) defined by Equation 3. Starting from the initial configuration corresponding to a sphere with a radius r = D max/2, where D max is the maximum diameter determined by the GNOM program, filled with small spheres (dummy atoms) with r 0 ≪ R, a simulated annealing algorithm is employed for the minimization procedure (35Svergun D.I. Biophys. J. 1999; 76: 2879-2886Abstract Full Text Full Text PDF PubMed Scopus (1756) Google Scholar). DAMMIN finally generates the best structure model containing a fraction of the initial small dummy atoms and also yields the SAXS intensity of the resulting model. The coordinate set of positions for E. coli GrpE was obtained from Protein Data Bank (1DKG) (17Harrison C.J. Hayer-Hartl M. Di Liberto M. Hartl F.U. Kuriyan J. Science. 1997; 276: 431-435Crossref PubMed Scopus (413) Google Scholar). Relative positions of the domains were found with an automated procedure that iteratively rotates their envelope functions to minimize the discrepancy with the ab initio low resolution structure. The models were displayed using the program MASSHA (36Konarev P.V. Petoukhov M.V. Svergun D.I. J. Appl. Crystallogr. 2001; 34: 527-532Crossref Scopus (130) Google Scholar). R g, maximum intraparticle distances (D max), envelope functions, and scattering curves were calculated from these atomic coordinates by the CRYSOL program taking into account the influence of the hydration shell (37Svergun D. Barberato C. Koch M.H. J. Appl. Crystallogr. 1995; 28: 768-773Crossref Scopus (2790) Google Scholar). SUPCOMB (38Kozin M.B. Svergun D.I. J. Appl. Crystallogr. 2001; 34: 33-41Crossref Scopus (1093) Google Scholar) was used to superimpose ab initio low resolution models onto crystallographic structures. The HydroPro program (39Garcia 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 R g, the sedimentation coefficient s, and tip-to-tip distance from the ab initio model generated by SAXS data at 20 °C. The HydroPro software was set up with radius of the atomic elements of 3.5 (from ab initio development), sigma factors from 5 to 8 (as indicate by supplier) and minibeads radius (SIGMIN and SIGMAX) from 6 to 2 Å after initial evaluation of the two extremes. The parameters Vbar, ρ, and η were estimated using the software Sednterp (www.jphilo.mailway.com/download.htm). The translational friction ratio or Perrin factor P, which indicates the relation of the frictional coefficient of the human GrpE particle to a sphere of the same molecular weight (f/f 0), was estimated by Solpro software (40Garcia de la Torre J. Carrasco B. Harding S.E. Eur. Biophys. J. 1997; 25: 361-372Crossref PubMed Scopus (65) Google Scholar). The SWISS-MODEL program (41Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9641) Google Scholar, 42Peitsch M.C. Biochem. Soc. Trans. 1996; 24: 274-279Crossref PubMed Scopus (900) Google Scholar, 43Peitsch M.C. Bio/Technology. 1995; 13: 658-660Crossref Scopus (116) Google Scholar) was used for modeling the human GrpE by homology with the crystal structure of E. coli GrpE (residues 34–197) (17Harrison C.J. Hayer-Hartl M. Di Liberto M. Hartl F.U. Kuriyan J. Science. 1997; 276: 431-435Crossref PubMed Scopus (413) Google Scholar) as the model for human GrpE48–191 (residues 48–191). The program PredictProtein (44Baldi P. Brunak S. Frasconi P. Pollastri G. Soda G. Bioinformatics. 1999; 15: 937-946Crossref PubMed Scopus (380) Google Scholar) was used to estimate the secondary structure of the N-terminal region (1–50 amino acids) of human GrpE, which was missing in the model generated by the SWISS-MODEL program. The N-terminal and its respective secondary structure prediction were loaded on software HyperChem (www.hallogram.com/index.html) for modeling and geometry optimization. This N-terminal model was then linked to the initial model (human Grp-E48–191) generated by SWISS-MODEL program and fitted in the bead model generated from SAXS data. After geometry optimization this whole model was analyzed in terms of tip-to-tip distance, radius of gyration, diffusion and sedimentation coefficients, and Perrin factor prediction using the HydroPro software simulating the same conditions as the experimental data were obtained. Cloning, Expression, and Purification—Human and E. coli GrpE amino acid sequences are shown in Fig. 1 for comparison, the two proteins have about 53% similarity and 30% identity. During the cloning of the human GrpE cDNA two sequence changes were made to allow recombinant expression of the functional protein in strain BL21(DE3). First, the cDNA 5′-sequence that codes for the mitochondrial peptide signal sequence was deleted. In human cells this peptide is removed from the protein when it reaches the mitochondria, but the bacterial host does not have the machinery to make this deletion. Without this modification, our expressed recombinant GrpE would have an N-terminal portion that is not present in the mitochondrial GrpE. This change causes the first residue in the mature protein to be leucine, which is not recognized as a start codon by strain BL21(DE3), thus the second change replaced this codon for a methionine codon. DNA sequencing confirmed the correct cloning and sequence of this mutated cDNA (data not shown). Human GrpE was expressed in a soluble state and in large amount, and the purification procedure resulted in a single protein band with the expected molecular mass (21.5 kDa) in SDS-PAGE (Fig. 2). Purified human GrpE shows no sign of impurities or degradation, is soluble (even in water), and folded (see below). Secondary Structure Analysis by CD and Thermal Analysis—CD spectropolarimetry was used to assess the protein secondary structure content as a function of temperature (Fig. 3, a and b). Analysis of the global shape of the far UV CD spectra indicates that human GrpE is folded and soluble, allowing measurements down to near 180 nm. The far UV CD spectrum shows characteristics of a highly α-helical protein, and analysis by the CDNN Deconvolution software indicates that about 55% of the protein is in this form (Fig. 3a). The CDNN software used the whole spectrum from 260 to 180 nm for secondary structure analysis, thus the calculation benefits from the good CD signal at low wavelengths. Increasing temperature causes the protein to unfold; there is a slight change in the CD signal at 222 nm from 4 to 50 °C and a more accentuated change above 50 °C (Fig. 3b). This temperature dependence is verified by DSC, the protein started to unfold at about 50 °C (Fig. 4). The unfolding of human GrpE is not completely reversible even if the protein is heated to only 50 °C as shown by both CD and DSC experiments (Figs. 3b and 4). The unfolding measured by DSC shows at least two unfolding events, one with a midpoint at
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