Reversible Unfolding of FtsZ Cell Division Proteins from Archaea and Bacteria
2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês
10.1074/jbc.m206723200
ISSN1083-351X
AutoresJosé M. Andreu, María A. Oliva, Octavio Monasterio,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoThe stability, refolding, and assembly properties of FtsZ cell division proteins from Methanococcus jannaschii and Escherichia coli have been investigated. Their guanidinium chloride unfolding has been studied by circular dichroism spectroscopy. FtsZ from E. coli and tubulin released the bound guanine nucleotide, coinciding with an initial unfolding stage at low denaturant concentrations, followed by unfolding of the apoprotein. FtsZ from M. jannaschiireleased its nucleotide without any detectable secondary structural change. It unfolded in an apparently two-state transition at larger denaturant concentrations. Isolated FtsZ polypeptide chains were capable of spontaneous refolding and GTP-dependent assembly. The homologous eukaryotic tubulin monomers misfold in solution, but fold within the cytosolic chaperonin CCT. Analysis of the extensive tubulin loop insertions in the FtsZ/tubulin common core and of the intermolecular contacts in model microtubules and tubulin-CCT complexes shows a loop insertion present at every element of lateral protofilament contact and at every contact of tubulin with CCT (except at loop T7). The polymers formed by purified FtsZ have a distinct limited protofilament association in comparison with microtubules. We propose that the loop insertions of tubulin and its CCT-assisted folding coevolved with the lateral association interfaces responsible for extended two-dimensional polymerization into microtubule polymers. The stability, refolding, and assembly properties of FtsZ cell division proteins from Methanococcus jannaschii and Escherichia coli have been investigated. Their guanidinium chloride unfolding has been studied by circular dichroism spectroscopy. FtsZ from E. coli and tubulin released the bound guanine nucleotide, coinciding with an initial unfolding stage at low denaturant concentrations, followed by unfolding of the apoprotein. FtsZ from M. jannaschiireleased its nucleotide without any detectable secondary structural change. It unfolded in an apparently two-state transition at larger denaturant concentrations. Isolated FtsZ polypeptide chains were capable of spontaneous refolding and GTP-dependent assembly. The homologous eukaryotic tubulin monomers misfold in solution, but fold within the cytosolic chaperonin CCT. Analysis of the extensive tubulin loop insertions in the FtsZ/tubulin common core and of the intermolecular contacts in model microtubules and tubulin-CCT complexes shows a loop insertion present at every element of lateral protofilament contact and at every contact of tubulin with CCT (except at loop T7). The polymers formed by purified FtsZ have a distinct limited protofilament association in comparison with microtubules. We propose that the loop insertions of tubulin and its CCT-assisted folding coevolved with the lateral association interfaces responsible for extended two-dimensional polymerization into microtubule polymers. chaperonia containing tailless polypeptide 1 guanidinium chloride dithiothreitol tris(2-carboxyethyl)phosphine hydrochloride 1,4-piperazinediethanesulfonic acid 4-morpholineethanesulfonic acid guanosine 5′-(α,β-methylenetriphosphate) The eukaryotic cytoskeletal proteins actin and tubulin probably have common ancestors with their respective prokaryotic homologs MreB (1van den Ent F. Amos L.A Löwe J. Nature. 2001; 413: 39-44Crossref PubMed Scopus (615) Google Scholar) and FtsZ (2Löwe J. Amos L.A. Nature. 1998; 391: 203-206Crossref PubMed Scopus (720) Google Scholar). MreB is a nucleotide-binding protein that is essential for bacterial rod shape (3Jones L.J.F. Carballido-Lopez R. Errington J. Cell. 2001; 104: 913-922Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar), whereas the GTP-binding protein FtsZ is the key component of the prokaryotic cell division machinery (4Rothfield L. Justice S. Garcia-Lara J. Annu. Rev. Genet. 1999; 33: 423-448Crossref PubMed Scopus (239) Google Scholar). Archaeal FtsZ from Methanococcus jannaschii (2Löwe J. Amos L.A. Nature. 1998; 391: 203-206Crossref PubMed Scopus (720) Google Scholar) and eukaryotic tubulin (5Nogales E. Wolf S.G. Downing K.H. Nature. 1998; 391: 199-203Crossref PubMed Scopus (1812) Google Scholar) share a common core of similarly folded N-terminal nucleotide-binding and middle structural domains. Their main differences include the tubulin C-terminal domain and several of the complex surface loops of tubulin; however, most of the seven nucleotide-binding loops are well conserved between FtsZ and tubulin, which constitute a distinct family of GTPases (6Nogales E. Downing K.H. Amos L.A. Löwe J. Nat. Struct. Biol. 1998; 5: 451-458Crossref PubMed Scopus (437) Google Scholar). No structure of a bacterial FtsZ has so far been reported. Tubulin αβ-dimers assemble into microtubules, long cylinders made of laterally associated protofilaments that form the mitotic spindle. Tubulin interacts with microtubule-associated proteins and with motor proteins mainly through its C-terminal domains. Docking the electron crystallographic structure of tubulin dimers (5Nogales E. Wolf S.G. Downing K.H. Nature. 1998; 391: 199-203Crossref PubMed Scopus (1812) Google Scholar, 7Löwe J., Li, H. Downing K.H. Nogales E. J. Mol. Biol. 2001; 313: 1045-1057Crossref PubMed Scopus (1005) Google Scholar) into lower resolution electron density maps of microtubules has generated pseudo-atomic models of microtubules (8Nogales E. Whittaker M. Milligan R.A. Downing K.H. Cell. 1999; 96: 79-88Abstract Full Text Full Text PDF PubMed Scopus (992) Google Scholar, 9Meurer-Grob P. Kasparian J. Wade R. Biochemistry. 2001; 40: 8000-8008Crossref PubMed Scopus (109) Google Scholar, 10Chacón P. Wriggers W. J. Mol. Biol. 2002; 317: 375-384Crossref PubMed Scopus (294) Google Scholar). On the other hand, FtsZ assembles at the future site of cell division, forming the so called Z-ring (11Bi E. Lutkenhaus J. Nature. 1991; 354: 161-164Crossref PubMed Scopus (1160) Google Scholar, 12Ben-Yehuda S. Losick R. Cell. 2002; 109: 257-266Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar), the structure of which has resisted observation. The Z-ring is stabilized by the binding of the C-terminal end of FtsZ to cell division proteins FtsA and ZipA; FtsA and ZipA recruit FtsK, which in turn interacts with the other septal proteins FtsQ, FtsL, FtsW, FtsI, and FtsN (4Rothfield L. Justice S. Garcia-Lara J. Annu. Rev. Genet. 1999; 33: 423-448Crossref PubMed Scopus (239) Google Scholar, 13Pichoff S. Lutkenhaus J. EMBO J. 2002; 21: 685-693Crossref PubMed Scopus (307) Google Scholar). FtsZ monomers form in vitro polymers made of tubulin-like protofilaments with the characteristic 4-nm axial spacing (14Erickson H.P. Taylor D.W. Taylor A.K. Bramhill D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 519-523Crossref PubMed Scopus (410) Google Scholar, 15Löwe J. Amos L.A. EMBO J. 1999; 18: 2364-2371Crossref PubMed Scopus (180) Google Scholar). Bacterial FtsZ expressed in mammalian cells does not co-assemble with microtubules, but can be induced to form other filaments (16Yu X.C. Margolin W. Gonzalez-Garay M.L. Cabral F. J. Cell Sci. 1999; 112: 2301-2311PubMed Google Scholar). It is conceivable that the structural complexity of tubulin relative to FtsZ has evolved together with its assembly into microtubules. Folding of many newly synthesized proteins in the cytosol is assisted by molecular chaperones (17Hartl F.U. Hayer-Hartl M. Science. 2002; 295: 1852-1858Crossref PubMed Scopus (2812) Google Scholar). Actin and tubulin are the paradigm of eukaryotic proteins that require the cytoplasmic chaperonin CCT1 (TriC) for theirin vivo or in vitro folding (18Gao Y. Thomas J.O. Chow R.L. Lee G.H. Cowan N.J. Cell. 1992; 69: 1043-1050Abstract Full Text PDF PubMed Scopus (425) Google Scholar, 19Yaffe M.B. Farr G.W. Miklos D. Horwich A.L. Sterlicht M.L. Sternlicht H. Nature. 1992; 358: 245-248Crossref PubMed Scopus (386) Google Scholar, 20Thulasiraman V. Yang C.F. Frydman J. EMBO J. 1999; 18: 85-95Crossref PubMed Scopus (269) Google Scholar, 21Shah C., Xu, C.Z. Vickers J. Williams R. Biochemistry. 2001; 40: 4844-4852Crossref PubMed Scopus (15) Google Scholar). On the other hand, the ease of overproducing soluble FtsZ and MreB in bacteria suggests that these proteins may be able to fold spontaneously. In fact, FtsZ was not identified as an in vivo substrate of the bacterial chaperonin GroEL (although it is a two-αβ-domain protein), but the less abundant cell division inhibitor MinD was (22Houry W.A. Frishman D. Eckerskorn C. Lottspeich F. Hartl U. Nature. 1999; 402: 147-154Crossref PubMed Scopus (436) Google Scholar); and FtsZ did not bind to GroEL or CCT in vitro (23Dobrzynski J.K. Sternlicht M.L. Peng I. Farr G.W. Sternlicht H. Biochemistry. 2000; 39: 3988-4003Crossref PubMed Scopus (19) Google Scholar). However, HscA (a protein of the Hsp70 family) and DnaK have been implicated in FtsZ ring formation through a chaperone-like activity (24Uehara T. Matsuzawa H. Nishimura A. Genes Cells. 2001; 6: 803-814Crossref PubMed Scopus (18) Google Scholar). Monomers of α- and β-tubulin bind to CCT in a quasi-native conformation in which the N-terminal, middle, and C-terminal domains are apparently opened up in the chaperonin cavity, and it has been proposed that the eukaryotic chaperonin coevolved with tubulin and actin (25Llorca O. Martin-Benito J. Ritco-Vonsovici M. Grantham J. Hynes G.M. Willison K. Carrascosa J. Valpuesta J.M. EMBO J. 2000; 19: 6971-6979Crossref Scopus (183) Google Scholar, 26Llorca O. Martin-Benito J. Gomez-Puertas P. Ritco-Vonsovici M. Willison K. Carrascosa J. Valpuesta J.M. J. Struct. Biol. 2001; 135: 205-218Crossref PubMed Scopus (57) Google Scholar). Upon ATP binding, CCT undergoes a structural change that closes the tubulin monomer into its native GTP-binding conformation, followed by ATP hydrolysis by CCT and tubulin release (27Llorca O. Martin-Benito J. Grantham J. Ritco-Vonsovici M. Willison K. Carrascosa J. Valpuesta J.M. EMBO J. 2001; 20: 4065-4075Crossref PubMed Scopus (117) Google Scholar). In this work, we have investigated the stability, folding, and assembly ability of refolded FtsZ molecules in comparison with tubulin. For this purpose, we first studied the unfolding by guanidinium chloride (GdmCl) of FtsZ from the hyperthermophilic archaeon M. jannaschii and from the bacterium Escherichia coli by circular dichroism spectroscopy. The unfolding of E. coli FtsZ and of tubulin from calf brain was multistage, starting with nucleotide release.M. jannaschii FtsZ unfolded in a single stage at higher GdmCl concentrations at 25 °C; however, it released its nucleotide at lower denaturant concentrations. The unfolding of FtsZ by GdmCl was reversible, yielding refolded FtsZ capable of GTP-dependent assembly, whereas the denaturation transitions of tubulin could not be reversed. We further analyzed the structures and assemblies of FtsZ and tubulin, which suggest that the loop insertions of tubulin and its CCT-assisted folding coevolved with the lateral association interfaces responsible for two-dimensional polymerization into microtubules. M. jannaschii FtsZ with a C-terminal Gly-Ser-His6 extension (M r 39,891, 372 residues) (2Löwe J. Amos L.A. Nature. 1998; 391: 203-206Crossref PubMed Scopus (720) Google Scholar) was overproduced in E. coli BL21(DE3) pLys and purified by HiTrap Ni2+ chelating affinity and Sephacryl S400 (Amersham Biosciences) size-exclusion chromatography. It typically contained ∼0.8 nucleotide bound, of which 80% was GDP and 20% was GTP; and it was stored concentrated (10–30 mg ml−1) at −70 °C (28Diaz J.F. Kralicek A. Mingorance J. Palacios J.M. Vicente M. Andreu J.M. J. Biol. Chem. 2001; 276: 17307-17315Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Its guanine nucleotide content was spectrophotometrically determined after extraction with cold 0.5n HClO4 employing an extinction coefficient of 12,400 m−1 cm−1 at 254 nm (29Correia J.J. Baty L.T. Williams R.C. J. Biol. Chem. 1987; 262: 17278-17284Abstract Full Text PDF PubMed Google Scholar). The concentration of M. jannaschii FtsZ was determined from its absorption spectrum in 6 m GdmCl, after subtraction of the absorbance due to the guanine nucleotide (ε254 = 13,620 m−1 cm−1 and ε280 = 8100 m−1cm−1 in 6 m GdmCl) (28Diaz J.F. Kralicek A. Mingorance J. Palacios J.M. Vicente M. Andreu J.M. J. Biol. Chem. 2001; 276: 17307-17315Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), employing apoprotein extinction coefficients of ε280 = 6970m−1 cm−1 and ε254 = 4275 m−1 cm−1 (calculated for 1 Trp, 1 Tyr, and 8 Phe residues) (30Edelhoch H. Biochemistry. 1967; 6: 1948-1954Crossref PubMed Scopus (3009) Google Scholar, 31). The approximate extinction coefficients determined for native M. jannaschii FtsZ are 11,600 m−1 cm−1 at 254 and 280 nm when containing 0.8 mol of nucleotide after dilution in neutral buffer and 13,000 m−1 cm−1 at 280 nm and 14,200−1 cm−1 at 254 nm when containing 1.0 mol of nucleotide typically after equilibration of the native protein in GDP- or GTP-containing buffer. M. jannaschii FtsZ without the unnatural Gly-Ser-His6 extension was constructed from the same plasmid, pHis17-mjFtsZ-H6 (2Löwe J. Amos L.A. Nature. 1998; 391: 203-206Crossref PubMed Scopus (720) Google Scholar), and purified by ammonium sulfate precipitation and ion-exchange and hydrophobic chromatography. 2M. A. Oliva, and J. M. Andreu, unpublished data. E. coli FtsZ (M r 40,324, 383 residues) was overproduced in E. coli BL21(DE3) and purified by two cycles of Ca2+-induced precipitation, followed by Mono Q anion-exchange chromatography (32Rivas G. Lopez A. Mingorance J. Ferrandiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). It typically contained 1.0 GDP bound per FtsZ and was stored concentrated (20–50 mg ml−1) at −70 °C. Its concentration was spectrophotometrically determined in 6 m GdmCl, after subtraction of the nucleotide absorption (as described above), employing extinction coefficients of ε280 = 3840 m−1 cm−1 and ε254 = 2750 m−1cm−1 (note that E. coli FtsZ has no Trp residues and 3 Tyr and 13 Phe residues). Bovine brain tubulin αβ-dimers were purified by ammonium sulfate precipitation, batch DEAE-Sephadex anion-exchange chromatography, and Mg2+-induced precipitation (33Lee J.C. Frigon R.P. Timasheff S.N. J. Biol. Chem. 1973; 248: 7253-7262Abstract Full Text PDF PubMed Google Scholar, 34Andreu J.M. Timasheff S.N. Biochemistry. 1982; 21: 6465-6476Crossref PubMed Scopus (149) Google Scholar, 35Andreu J.M. Perez-Ramirez B. Gorbunoff M.J. Ayala D. Timasheff S.N. Biochemistry. 1998; 37: 8356-8368Crossref PubMed Scopus (76) Google Scholar) and stored concentrated (∼100 mg ml−1) under liquid nitrogen. Tubulin αβ-dimers have 896 residues and a molecular weight of 99,929, calculated for the major tubulin isotypes from pig brain, not taking into account other isotypes and post-translational modifications (for review, see Ref. 36Ludueña R.F. Int. Rev. Cytol. 1998; 178: 207-275Crossref PubMed Google Scholar). Tubulin contained 1.6–2.0 mol of bound GTP. Tubulin dimer concentration was spectrophotometrically determined at 276 nm employing the previously measured extinction coefficients of 109,000m−1 cm−1 in 6 m GdmCl (the value calculated as described above for 8 Trp and 35 Tyr residues and 1.8 ± 0.2 GTP is 109,000 ± 2000 M−1cm−1) and 116,000 m−1cm−1 in neutral buffer after light scattering correction (34Andreu J.M. Timasheff S.N. Biochemistry. 1982; 21: 6465-6476Crossref PubMed Scopus (149) Google Scholar). GdmCl and dithiothreitol (DTT) were from Calbiochem (Ultrol grade), and TCEP was from Interchim. Pipes, Hepes, Mes, and GDP were from Sigma. GTP (lithium salt) was from Roche Molecular Biochemicals. Other analytical grade chemicals were from Merck. CD spectra were acquired with Jasco 720 and 810 spectropolarimeters employing 1-mm cells in thermostatted cell holders (0.1- and 0.2-mm cells for spectra below 210 nm). The temperature was measured with a small thermocouple placed into the cells. Four scans of each sample or buffer (1-nm bandwidth and measurement interval, 20 nm min−1 scan speed, and 4-s time constant) were averaged, but not smoothed. Accurate CD measurements at fixed wavelengths were made from 5- to 10-min time recordings of each sample (10-s intervals and 16-s time constant). CD data (millidegrees) were reduced to mean residue ellipticity values (degrees cm2 dmol−1) with Jasco J700 and J800 software and plotted with SigmaPlot. Fluorescence measurements were made with a Fluorolog 3-221 instrument (Jobin Yvon-Spex, Longiumeau, France) employing 2-nm excitation and 5-nm emission bandwidths. Solutions of GdmCl were prepared gravimetrically, and their concentration was confirmed by refractometry (37Pace C.N. Scholtz J.M. Creighton T.E. Protein Structure, a Practical Approach. 2nd Ed. Oxford University Press, Oxford1997: 299-321Google Scholar). Concentrated protein stock solutions were diluted into 6 or 8m GdmCl and 5 mm DTT solutions in buffer. Alternately, more concentrated FtsZ solutions in 6 m GdmCl were prepared by gravimetric addition of 1.009 mg of GdmCl/μl of protein stock. Proteins were held a minimum of 0.5 h in 6m GdmCl at 25 °C before refolding. For protein refolding, the denaturant concentration was reduced by dialysis (4 °C, >16 h) or by dilution (50- or 100-fold) in buffer. Buffers were 20 mm Pipes-KOH and 5 mm DTT (pH 7.5) for FtsZ and 10 mm sodium phosphate and 5 mm DTT (pH 7.0) for tubulin. Equilibrium GdmCl unfolding and refolding curves (37Pace C.N. Scholtz J.M. Creighton T.E. Protein Structure, a Practical Approach. 2nd Ed. Oxford University Press, Oxford1997: 299-321Google Scholar, 38Eftink M. Methods Enzymol. 1995; 259: 487-512Crossref PubMed Scopus (74) Google Scholar) were determined by careful dilution of native and denatured proteins, respectively, into the corresponding GdmCl solutions in the same buffers with 5 mm DTT, followed by equilibration (in an Eppendorf Thermostat Plus) and CD measurements at 5 ± 1, 25 ± 1, and 42 ± 1 °C. At 25 °C, M. jannaschiiFtsZ required a minimum of 36 h to reach equilibrium in the transition GdmCl range in both directions (but only a few minutes at 0 and 6 m GdmCl), whereas E. coli FtsZ required 2 h and tubulin required 1 h for equilibration at intermediate GdmCl concentrations. Measurements of GdmCl-induced unfolding of M. jannaschii FtsZ at 55 ± 1 and 70 ± 1 °C were made by directly taking CD time recordings until attainment of equilibrium (<100 min at 55 °C and 5 m GdmCl (Fig. 1 A, dashed line). Remarkably, when the denaturant was dialyzed in the cold, M. jannaschii FtsZ recovered a CD spectrum indistinguishable from the native spectrum (Fig. 1 A, circles and solid line, respectively). Similar results were obtained with 10 μmGDP, 10 μm GTP, or without nucleotide in the dialysis buffer; these dialyzed samples contained 1.0 mol (GDP), 0.8 (GTP), and 0.5 (no addition) of nucleotide/FtsZ, respectively. Similar CD results were also obtained upon 50-fold dilution of FtsZ solutions in 6m GdmCl into buffer at 2, 25, or 53 °C (data not shown). At 80 °C, there was a 20% decrease in the CD value at 222 nm without denaturant with respect to the value at 25 °C, suggesting a partial thermal unfolding of the protein. At this high temperature,M. jannaschii FtsZ partially refolded upon dilution to an extent of ∼45% from a comparison with the CD values of the controls at 222 nm. Determination of the equilibrium GdmCl unfolding-refolding curve ofM. jannaschii FtsZ at 25 °C from the CD data at 222 nm indicated a single-stage transition between ∼2.2 and 4 mGdmCl, which was fully reversible within experimental error and had a [GdmCl] 12 (midpoint) at 3.1 m GdmCl (Fig.1 B). There is no evidence for any residual structure above 5m GdmCl. The curve could be fitted by a two-state equilibrium model (Fig. 1 B, solid line), suggesting (but not proving) that both domains of M. jannaschii FtsZ unfold simultaneously. Deletion of the unnatural C-terminal Gly-Ser-His6 extension from the M. jannaschii FtsZ protein had no significant effect on the GdmCl unfolding-refolding curve. The [GdmCl] 12 values determined at three other temperatures from the change in the CD value at 222 nm were as follows: 5 °C, 2.3 m; 55 °C, 2.8m; and 70 °C, 2.2 m (Fig. 1 B,inset). This indicates that M. jannaschii FtsZ is maximally stabilized at ∼40 °C, and it is less stable in the cold and at higher temperatures; from the trend of the data, FtsZ may be marginally stable at the 85 °C optimal growth temperature ofM. jannaschii (41Jones W.J. Leigh J.A. Mayer F. Woese C.R. Wolfe R.S. Arch. Microbiol. 1983; 136: 254-261Crossref Scopus (431) Google Scholar). Monitoring the unfolding of M. jannaschii FtsZ by the change in fluorescence emission intensity of its single tryptophan (Trp319, which is in the second domain, roughly diametrically opposite of the nucleotide-binding site) indicated a similar transition centered at 3.2 m GdmCl (data not shown). However, the release of the nucleotide bound to FtsZ, measured in the solution after sedimenting the protein, took place at lower denaturant concentrations (midpoint of ∼1.5 m GdmCl), at which the CD change was practically negligible (nucleotide release was confirmed by chromatography in Sephadex G-25 columns equilibrated in 1 and 2 m GdmCl). This indicates that the average secondary structure of M. jannaschii FtsZ at 25 °C is practically insensitive to GDP binding and that the unfolding measured by CD spectroscopy is that of the apoprotein. However, the nucleotide binding must further stabilize the native protein, and it is known to induce local structural changes in M. jannaschii FtsZ (28Diaz J.F. Kralicek A. Mingorance J. Palacios J.M. Vicente M. Andreu J.M. J. Biol. Chem. 2001; 276: 17307-17315Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar).M. jannaschii FtsZ in 6 m GdmCl could be separated from the nucleotide by chromatography on Sephadex G-25 (Fig.2, curves a; 0.9 mol of nucleotide released per FtsZ). However, when unfolded M. jannaschii FtsZ was diluted from 6 to 0.12 m GdmCl in the cold and chromatographed on a column without denaturant, the protein bound again ∼80% of its released nucleotide (Fig. 2,curves b; 0.2 mol of nucleotide released per FtsZ), even in this experiment carried out in the absence of added nucleotide. This indicate that GdmCl-unfolded M. jannaschii FtsZ can easily regain functionality. E. coliFtsZ unfolded in GdmCl (Fig.3 A, dashed line), and it refolded upon dilution of the denaturant in 20 mmPipes and 5 mm DTT (pH 7.5) at 25 °C, as judged from the CD spectra, in which 93–97% of the 222 nm ellipticity of the native protein was typically recovered (Fig. 3 A, open circles and solid line, respectively). A similar result was obtained in 100 mm potassium glutamate, 200 mm potassium acetate, 5 mm magnesium acetate, and 20 mm Hepes-KOH (pH 7.5) (potassium glutamate-acetate buffer), resembling the physiological osmolytes in the E. coli cytoplasm (42Record Jr., M.T. Courtenay E.S. Cayley S. Guttman H.J. Trends Biochem. Sci. 1998; 23: 190-194Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) (within the experimental error from subtracting the dichroism of l-glutamate). The spectrum obtained by dilution in ice-cold E. coli FtsZ assembly buffer containing 5 mm DTT (pH 6.5) matched the CD spectrum of the native control. However, complete refolding ofE. coli FtsZ was not obtained by dialysis (in Pipes/DTT orE. coli FtsZ assembly buffer/DTT with 10 μmGTP), in which this protein partially refolded and lost most of the
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