Thermal Stability and Aggregation of Sulfolobus solfataricus β-Glycosidase Are Dependent upon the N-∈-Methylation of Specific Lysyl Residues
2004; Elsevier BV; Volume: 279; Issue: 11 Linguagem: Inglês
10.1074/jbc.m308520200
ISSN1083-351X
AutoresFerdinando Febbraio, Annapaola Andolfo, Fabio Tanfani, Raffaella Briante, Fabrizio Gentile, Silvestro Formisano, C. Vaccaro, Andrea Scirè, Enrico Bertoli, Piero Pucci, Roberto Nucci,
Tópico(s)Enzyme Structure and Function
ResumoMethylation in vivo is a post-translational modification observed in several organisms belonging to eucarya, bacteria, and archaea. Although important implications of this modification have been demonstrated in several eucaryotes, its biological role in hyperthermophilic archaea is far from being understood. The aim of this work is to clarify some effects of methylation on the properties of β-glycosidase from Sulfolobus solfataricus, by a structural comparison between the native, methylated protein and its unmethylated counterpart, recombinantly expressed in Escherichia coli. Analysis by Fourier transform infrared spectroscopy indicated similar secondary structure contents for the two forms of the protein. However, the study of temperature perturbation by Fourier transform infrared spectroscopy and turbidimetry evidenced denaturation and aggregation events more pronounced in recombinant than in native β-glycosidase. Red Nile fluorescence analysis revealed significant differences of surface hydrophobicity between the two forms of the protein. Unlike the native enzyme, which dissociated into SDS-resistant dimers upon exposure to the detergent, the recombinant enzyme partially dissociated into monomers. By electrospray mapping, the methylation sites of the native protein were identified. A computational analysis of β-glycosidase three-dimensional structure and comparisons with other proteins from S. solfataricus revealed analogies in the localization of methylation sites in terms of secondary structural elements and overall topology. These observations suggest a role for the methylation of lysyl residues, located in selected domains, in the thermal stabilization of β-glycosidase from S. solfataricus. Methylation in vivo is a post-translational modification observed in several organisms belonging to eucarya, bacteria, and archaea. Although important implications of this modification have been demonstrated in several eucaryotes, its biological role in hyperthermophilic archaea is far from being understood. The aim of this work is to clarify some effects of methylation on the properties of β-glycosidase from Sulfolobus solfataricus, by a structural comparison between the native, methylated protein and its unmethylated counterpart, recombinantly expressed in Escherichia coli. Analysis by Fourier transform infrared spectroscopy indicated similar secondary structure contents for the two forms of the protein. However, the study of temperature perturbation by Fourier transform infrared spectroscopy and turbidimetry evidenced denaturation and aggregation events more pronounced in recombinant than in native β-glycosidase. Red Nile fluorescence analysis revealed significant differences of surface hydrophobicity between the two forms of the protein. Unlike the native enzyme, which dissociated into SDS-resistant dimers upon exposure to the detergent, the recombinant enzyme partially dissociated into monomers. By electrospray mapping, the methylation sites of the native protein were identified. A computational analysis of β-glycosidase three-dimensional structure and comparisons with other proteins from S. solfataricus revealed analogies in the localization of methylation sites in terms of secondary structural elements and overall topology. These observations suggest a role for the methylation of lysyl residues, located in selected domains, in the thermal stabilization of β-glycosidase from S. solfataricus. Post-translational modifications expand the cellular repertoire of proteins far beyond the possibilities offered by the 20 encoded amino acids, constituting one of the most exciting frontiers in modern biology. A little more than a dozen post-translational protein side-chain modifications have been identified so far. Protein methylation occurs ubiquitously in nature, in bacteria, archaea, and eucarya, involving the amino groups of such residues as arginine, lysine, histidine, alanine, proline, and glutamine and hydroxyl groups of glutamic and aspartic acid (1Paik W.K. Kim S. Yonsei Med. J. 1986; 27: 159-177Crossref PubMed Scopus (5) Google Scholar). Methylated amino acids occur in highly specialized proteins, exerting diverse functional and structural roles, such as histones, flagellar proteins, myosin, actin, ribosomal proteins, opsin, EF-1α, HnPNP protein, HMG-1 and -2 proteins, fungal and plant cytochrome c, myelin basic protein, EF-Tu, heat shock proteins, calmodulin, etc. (1Paik W.K. Kim S. Yonsei Med. J. 1986; 27: 159-177Crossref PubMed Scopus (5) Google Scholar). The effects of methylation on the recruitment of heterochromatin proteins to specific histones in nucleosome cores and the subsequent effects on gene expression have been documented in detail (2Dillon N. Festenstein R. Trends Genet. 2002; 18: 252-258Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). It was reported that methylation of the histone H3 tail is an epigenetic mark, affecting acetylation and phosphorylation of histone tail residues in mammals and Drosophila (3Rea S. Eisenhaber F. O'Carroll D. Strahl B.D. Sun Z.W. Schmid M. Opravil S. Mechtler K. Ponting C.P. Allis C.D. Jenuwein T. Nature. 2000; 406: 593-599Crossref PubMed Scopus (2199) Google Scholar, 4Czermin B. Schotta G. Hulsmann B.B. Brehm A. Becker P.B. Reuter G. Imhof A. EMBO Rep. 2001; 2: 915-919Crossref PubMed Scopus (146) Google Scholar). A model was suggested, in which the concerted deacetylation and methylation of Lys9 of histone H3 led to a permanent silencing of transcription in particular areas of the genome (4Czermin B. Schotta G. Hulsmann B.B. Brehm A. Becker P.B. Reuter G. Imhof A. EMBO Rep. 2001; 2: 915-919Crossref PubMed Scopus (146) Google Scholar). Methylation created a high affinity binding site for heterochromatin protein 1 (5Lachner M. O'Carroll D. Rea S. Mechtler K. Jenuwein T. Nature. 2001; 410: 116-120Crossref PubMed Scopus (2193) Google Scholar, 6Bannister A.J. Zegerman P. Partridge J.F. Miska E.A. Thomas J.O. Allshire R.C. Kouzarides T. Nature. 2001; 410: 120-124Crossref PubMed Scopus (2196) Google Scholar), which dimerized and thereby promoted the formation of higher order structures (7Nielsen A.L. Oulad-Abdelghani M. Ortiz J.A. Remboutsika E. Chambon P. Losson R. Mol. Cell. 2001; 7: 729-739Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). In addition, histone methylation affected DNA methylation in the heterochromatin of Neurospora crassa (8Tamaru H. Selker E.U. Nature. 2001; 414: 277-283Crossref PubMed Scopus (862) Google Scholar). The essential role of lysine methylation was demonstrated also in rat ribosomal proteins (9Williamson N.A. Raliegh J. Morrice N.A. Wettenhall R.E. Eur. J. Biochem. 1997; 246: 786-793Crossref PubMed Scopus (23) Google Scholar) and in the pathogenesis of late infantile ceroid lipofuscinosis (10Katz M.L. Siakotos A.N. Gao Q. Freiha B. Chin D.T. Biochim. Biophys. Acta. 1997; 1361: 66-74Crossref PubMed Scopus (13) Google Scholar). A recent observation of high medical interest concerns the inhibition of toxicity of the synthetic β-amyloid peptide β-(25–35) by N-methylation (11Hughes E. Burke R.M. Doig A.J. J. Biol. Chem. 2000; 275: 25109-25115Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). The presence of methylated lysyl residues has also been reported for proteins from thermophilic microorganisms, such as ferredoxin from the thermoacidophilic archaeon Sulfolobus acidocaldarius (12Minami Y. Wakabayashi S. Wada K. Matsubara H. Kerscher L. Oesterhelt D. J. Biochem. (Tokyo). 1985; 97: 745-753Crossref PubMed Scopus (59) Google Scholar), and a number of proteins from the hyperthermophilic archaeon Sulfolobus solfataricus (13Ammendola B. Raia C.A. Caruso C. Camardella L. D'Auria S. De Rosa M. Rossi M. Biochemistry. 1992; 31: 12514-12523Crossref PubMed Scopus (91) Google Scholar, 14Maras B. Consalvi V. Chiaraluce R. Politi L. De Rosa M. Bossa F. Scandurra R. Barra D. Eur. J. Biochem. 1992; 203: 81-87Crossref PubMed Scopus (70) Google Scholar, 15Zappacosta F. Sannia G. Savoy L.A. Marino G. Pucci P. Eur. J. Biochem. 1994; 222: 761-767Crossref PubMed Scopus (18) Google Scholar). In this regard, it has been shown that the rate constant of thermoin-activation of an enzyme is an inverse function of the number of modified ∈-amino groups (16Torchilin V.P. Maksimenko A.V. Smirnov V.N. Klibanov A.M. Martinek K. Biochim. Biophys. Acta. 1979; 567: 1-11Crossref PubMed Scopus (66) Google Scholar). However, no systematic study of the effect and nature of N-∈-methylation on the structural and catalytic properties of thermophilic enzymes has been carried out. β-Glycosidase from S. solfataricus (Ssβgly) 1The abbreviations used are: Ssβgly, S. solfataricus β-glycosidase; EcSsβgly, S. solfataricus β-glycosidase recombinantly expressed in E. coli; SsADH, S. solfataricus alcohol dehydrogenase; FT-IR, Fourier transform infrared; HPLC, high performance liquid chromatography; ES-MS, electrospray mass spectrometry; MALDI, matrix-assisted laser desorption ionization; amide I′, amide I band in a [2H]2O medium; ES, electrospray; MS, mass spectrometry. has been recently subjected to a detailed analysis of the structural determinants of protein stability, prompted by its peculiar behavior in the presence of SDS (17Gentile F. Amodeo P. Febbraio F. Picaro F. Motta A. Formisano S. Nucci R. J. Biol. Chem. 2002; 277: 44050-44060Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). This protein may also be adequate as a model for investigating the structural and functional consequences of N-∈-methylation of lysyl residues in archaea. In the present study, a detailed structural characterization of the native, methylated Ssβgly and the unmethylated enzyme, recombinantly expressed in Escherichia coli (EcSsβgly) (18Moracci M. Nucci R. Febbraio F. Vaccaro C. Vespa N. La Cara F. Rossi M. Enzyme Microb. Technol. 1995; 17: 992-997Crossref PubMed Scopus (70) Google Scholar), was carried out. In order to discriminate between the different behaviors of the methylated and unmethylated forms of the protein, their thermal denaturation was investigated by Fourier transform infrared (FT-IR) spectroscopy and turbidimetry. The effects of methylation on protein stability in SDS were also investigated. Methylated residues were localized within the sequence by mass spectrometry. Moreover, the localization of methylation sites was analyzed in relation with neighboring elements of secondary structure and with the overall topology of the enzyme and also in relation with the pattern of sequence conservation between Ssβgly and other mesophilic β-glucosidases belonging to glycosyl hydrolase family I. Materials—S. solfataricus strain MT4, isolated from acidic hot springs at Pozzuoli (Naples), was grown aerobically at 87 °C and pH 3.0 in a 100-liter fermenter, as previously described (19Cacace M.G. De Rosa M. Gambacorta A. Biochemistry. 1976; 15: 1692-1696Crossref PubMed Scopus (10) Google Scholar). Homogeneous Ssβgly (20Nucci R. Moracci M. Vaccaro C. Vespa N. Rossi M. Biotechnol. Appl. Biochem. 1993; 17: 239-250PubMed Google Scholar) and EcSsβgly (18Moracci M. Nucci R. Febbraio F. Vaccaro C. Vespa N. La Cara F. Rossi M. Enzyme Microb. Technol. 1995; 17: 992-997Crossref PubMed Scopus (70) Google Scholar) were purified as described. Cyanogen bromide was purchased from Pierce. Trypsin was from Sigma, and endoproteases Glu-C, Asp-N, and Lys-C were obtained from Roche Molecular Biochemicals. Deuterium oxide (99.9%, [2H]2O) was purchased from Aldrich. All other reagents and solvents were of the highest purity. The high pressure liquid chromatography (HPLC) systems were from Hewlett Packard and Waters. The Nile Red and the standard of N-∈-methyl-l-lysine were from Sigma. The AccQ·Tag kit for amino acid analysis was from Waters. Amino Acid Composition Analysis—Aliquots of homogeneous samples of Ssβgly were desalted by reverse phase HPLC on a Sephasil C4 (5-μm) column and lyophilized in pyrolyzed glass tubes at 500 °C. Amino acid composition was determined on samples hydrolyzed in 6 n HCl at 153 °C for 1 h in sealed vials under vacuum. The sample was lyophilized and suspended in 20 mm HCl and derivatized using the AccQ·Fluor reagent kit (Waters), as described in the AccQ·Tag Method (Waters). Aliquots of derivatized samples were injected in a Breeze HPLC system (Waters) on an AccQ·Tag amino acid analysis (high efficiency Nova-Pak™ C18; 4 μm) column (Waters), specifically certified for use with the AccQ·Tag method. The elution gradient in the AccQ·Tag Method was slightly modified in order to separate peaks of derivatized standards of l-leucine (retention time 33.3 min) and N-∈-methyl-l-lysine (retention time 33.6 min). Preparation of Samples for Infrared Measurements—Typically, 1–1.5 mg of homogeneous native or recombinant protein, dissolved in the buffer used for their purification (20Nucci R. Moracci M. Vaccaro C. Vespa N. Rossi M. Biotechnol. Appl. Biochem. 1993; 17: 239-250PubMed Google Scholar), were centrifuged in 30K centricon microconcentrators (Amicon) at 3000 × g and 4 °C and concentrated to a volume of ∼40 μl. Then 300 μl of 50 mm phosphate buffer, p2H 7.0, in [2H]2O, were added, and the samples were concentrated again. The p2H value corresponds to the pH meter reading + 0.4 (21Salomaa P. Schaleger L.L. Long F.A. J. Am. Chem. Soc. 1964; 86: 1-7Crossref Scopus (288) Google Scholar). This procedure was repeated several times in order to replace completely the original buffer with the phosphate buffer. In the last washing, the protein samples were concentrated to a final volume of 40 μl and used for the infrared measurements. The time of contact of the proteins with the [2H]2O medium prior to FT-IR analysis was about 24 h. At least three different measurements were carried out on different samples of each protein. Infrared Spectra—The concentrated homogeneous protein samples were placed in a thermostatted Graseby Specac 20500 cell (Graseby-Specac Ltd., Orpington, Kent, UK) fitted with CaF2 windows and 25-μm Teflon spacers. FT-IR spectra were recorded by means of a PerkinElmer Life Sciences 1760-x Fourier transform infrared spectrometer, using a deuterated triglycine sulfate detector and a normal Beer-Norton apodization function. At least 24 h before and during data acquisition, the spectrometer was continuously purged with dry air at a dew point of –40 °C. Spectra of buffers and samples were acquired at 2-cm–1 resolution, under the same scanning and temperature conditions. Typically, 256 scans were averaged for each spectrum obtained at 20 °C, whereas 32 scans were averaged for spectra obtained at higher temperatures. In the thermal denaturation experiments, the temperature was raised by 5 ± 0.1 °C steps from 20 to 95 °C. Before acquiring spectra, samples were maintained at the desired temperature for the time necessary for the stabilization of temperature inside the cell (6 min). Spectra were collected and processed using the Spectrum software from PerkinElmer. The deconvoluted parameters for the amide I band were set with a γ value of 2.5 and a smoothing length of 60. Turbidimetric Measurements—Samples containing 250 μg/ml homogeneous Ssβgly or EcSsβgly in 50 mm sodium phosphate buffer, pH 7.0, were filtered on 0.22-μm sterile filters (Millipore Corp.). Absorbances were recorded in a 1-cm light path quartz cuvette, at a wavelength of 600 nm, using a spectrophotometer Cary 1E thermostatted with a Cary temperature controller accessory, equipped with a Peltier's heat exchange device positioned around the sample, with an error of 0.1 °C. The temperature was increased from 50 to 95 °C at a rate of 1 °C/min, and the increase in absorbance at 600 nm was recorded. At least three different measurements were carried out on different samples and at different concentrations. Thermal Stability Measurements—The thermal stability of Ssβgly and EcSsβgly was measured by incubating a 0.41 μm concentration of the homogeneous enzyme in 0.1 m sodium phosphate buffer, pH 6.5, at 85 °C in a thermostatted water bath. At time intervals, aliquots containing 4.1 pmol of the enzyme were withdrawn from the incubation mixture and assayed at 75 °C under the conditions described (20Nucci R. Moracci M. Vaccaro C. Vespa N. Rossi M. Biotechnol. Appl. Biochem. 1993; 17: 239-250PubMed Google Scholar). Nile Red Fluorescence—All analyses were performed with a Jasco FP777 spectrofluorimeter, thermostatted at room temperature, using cells with a working volume of 500 μl and a path length of 10 mm. The excitation wavelength was set at 550 nm, and excitation and emission slits were set at 5 and 10 nm, respectively. Nile Red (0.25 mm in Me2SO) was added in the cuvette containing homogeneous samples of 0.41 μm Ssβgly or EcSsβgly, up to a final concentration of 1 μm, and spectra were recorded after 15 min. In a hydrophobic environment, a blue shift in the maximum emission wavelength (about 665 nm in water) and an increase in the fluorescence intensity of Nile Red are observed. Preparation of Linear Transverse Gradient Polyacrylamide Gels— The preparation of linear transverse-gradient polyacrylamide gels was described in detail (17Gentile F. Amodeo P. Febbraio F. Picaro F. Motta A. Formisano S. Nucci R. J. Biol. Chem. 2002; 277: 44050-44060Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Linear transverse gradient gels were used, containing 4–9% total acrylamide from left to right, 2.7% N,N′-methylene bis-acrylamide, in 0.375 m Tris/HCl, pH 8.8, 0.1% SDS, polymerized on a sheet of GelBond-PAG (Bio-Whittaker). No stacking gel was used, but a sample gel containing 3.75% total acrylamide in electrode buffer was created on top of the gel. Reference standards and Ssβgly or EcSsβgly were subjected to electrophoresis all together in alternate lanes of 20-lane gels. The electrode buffer contained 0.025 m Tris base, 0.19 m glycine, pH 8.2, 0.1% SDS. The enzymes were dissolved in 0.01 m Tris/HCl, pH 6.8, 1% SDS, 0.7 m β-mercaptoethanol, 1.36 m glycerol, and 0.005% bromphenol blue as tracking dye. SDS electrophoresis was conducted at 15 °C at 10 mA, until the dye front reached the lower end on the left side of the gels. The gels were stained with Coomassie Brilliant Blue R-250. Molecular Mass Estimation by Ferguson Analysis of Linear Transverse Gradient Polyacrylamide Gels—The molecular mass of Ssβgly and EcSsβgly was determined as described (17Gentile F. Amodeo P. Febbraio F. Picaro F. Motta A. Formisano S. Nucci R. J. Biol. Chem. 2002; 277: 44050-44060Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), by indirect comparison of the relative mobilities (Rm) of the enzyme and those of calibration proteins, after electrophoresis in a linear transverse gradient polyacrylamide gel. A detailed description of the method was reported (17Gentile F. Amodeo P. Febbraio F. Picaro F. Motta A. Formisano S. Nucci R. J. Biol. Chem. 2002; 277: 44050-44060Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Chemical and Enzymatic Hydrolyses—Homogeneous β-glycosidase samples were digested by CNBr in 70% trifluoroacetic acid, at room temperature, for 18 h in the dark. Samples were diluted with 4 volumes of distilled H2O and lyophilized. CNBr fragments were purified by HPLC on a Vydac C4 column (25 × 0.46 cm; 5 μm), using 0.1% trifluoroacetic acid (solvent A) and 0.07% trifluoroacetic acid in 95% acetonitrile (solvent B), by means of a two-step gradient. The column was equilibrated at 20% of solvent B for 5 min, and then the acetonitrile concentration was raised from 20 to 35% in 15 min and from 35 to 60% in 33 min. The elution was monitored at 220 and 280 nm. Selected CNBr peptides from Ssβgly were subdigested overnight at 37 °C with endoproteinase Asp-N in 0.4% ammonium bicarbonate containing 10% acetonitrile, pH 8.5, using an enzyme-to-substrate ratio of 1:100 (w/w). Subdigestion of the peptide mixtures by endoproteinase Glu-C, trypsin, or endoproteinase Lys-C was carried out in 50 mm ammonium bicarbonate, pH 8.5, for 18 h at 37 °C, using an enzyme-tosubstrate ratio of 1:50 (w/w). Under the alkaline conditions used for the enzymatic digestions, the C-terminal lactone of each CNBr fragment was hydrolyzed to free homoserine. Samples were then lyophilized twice prior to matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Mass Spectrometry—Intact homogeneous proteins or individual peptide fractions were submitted to electrospray mass spectrometry (ES-MS), using a BIO-Q triple quadrupole mass spectrometer (Micromass). Samples were dissolved in 1% acetic acid in 50% acetonitrile, and 2–10 μl were injected into the mass spectrometer at a flow rate of 10 μl/min. The quadrupole was scanned from m/z 600 to 1800 at 10 s/scan, and the spectra were acquired and elaborated using the MassLynx software (Micromass). Calibration was performed with the multiply charged ions from a separate injection of myoglobin (Mr = 16,951.5). All mass values are reported as average masses. MALDI mass spectra were recorded using a Voyager DE MALDI-time-of-flight mass spectrometer (Applied Biosystems). A mixture of analyte solution, α-cyano-4-hydroxycinnamic acid, and bovine insulin was applied to the sample plate and air-dried. Mass calibration was performed using the molecular ions from bovine insulin at 5734.6 and a matrix peak at 379.1 as internal standards. Raw data were analyzed with a computer software provided by the manufacturer and were reported as average masses. Computational Analysis—All structure calculations were performed on a SGI IRIS O2 R10000 computer. Our simulations were based on the crystal structure of Ssβgly refined at 2.6 Å (Protein Data Bank entry 1GOW) (22Aguilar C.F. Sanderson I. Moracci M. Ciaramella M. Nucci R. Rossi M. Pearl L.H. J. Mol. Biol. 1997; 271: 789-802Crossref PubMed Scopus (225) Google Scholar). Lysines 116, 135, 273, 311, and 332 were modified, using the Biopolymer module implemented in InsightII 98.0 (Biosym/MSI) (23Biosym/MSI InsightII User Guide. Biosym/MSI, San Diego, CA1995Google Scholar), by the addition of a methyl group, from the InsightII library, to the side chain N∈ atom. In the same module, hydrogen atoms were added to the methylated and unmethylated protein structures at pH 7.0. The cvff force field was applied to the two structures, and the resulting protonated state of the modified and unmodified protein gave a total charge of –36. The positions of the side chains were optimized by subsequent molecular mechanics calculations, using the Discover 3 module implemented in InsightII 98.0 (Biosym/MSI) (23Biosym/MSI InsightII User Guide. Biosym/MSI, San Diego, CA1995Google Scholar). For the minimization process, a conjugate gradient was applied after 50 steps of steepest descent algorithm. After minimization, the differences in the N-∈-methyl-lysine area were analyzed by the Viewer module of InsightII 98.0. The exposed solvent surfaces of the modified and unmodified proteins were calculated using the Molmol program, release 2.5 (24Koradi R. Billeter M. Wûthrich K. J. Mol. Graphics. 1996; 14: 51-55Crossref PubMed Scopus (6498) Google Scholar), with a standard radius of 1.4 Å. Comparisons between the sequences of β-glycosidase (Swiss-Prot accession number P22498) (25Cubellis M.V. Rozzo C. Montecucchi P. Rossi M. Gene (Amst.). 1990; 94: 89-94Crossref PubMed Scopus (88) Google Scholar), alcohol dehydrogenase (SsADH) (Swiss-Prot accession number P39462) (13Ammendola B. Raia C.A. Caruso C. Camardella L. D'Auria S. De Rosa M. Rossi M. Biochemistry. 1992; 31: 12514-12523Crossref PubMed Scopus (91) Google Scholar), aspartate aminotransferase (pir accession number S07088) (26Cubellis M.V. Rozzo C. Nitti G. Arnone M.I. Marino G. Sannia G. Eur. J. Biochem. 1989; 186: 375-381Crossref PubMed Scopus (60) Google Scholar), and glutamate dehydrogenase (pir accession number S20286) (14Maras B. Consalvi V. Chiaraluce R. Politi L. De Rosa M. Bossa F. Scandurra R. Barra D. Eur. J. Biochem. 1992; 203: 81-87Crossref PubMed Scopus (70) Google Scholar) from S. solfataricus were performed using ClustalX (27Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56002) Google Scholar). Structure comparisons between Ssβgly (Protein Data Bank accession number 1GOW) (22Aguilar C.F. Sanderson I. Moracci M. Ciaramella M. Nucci R. Rossi M. Pearl L.H. J. Mol. Biol. 1997; 271: 789-802Crossref PubMed Scopus (225) Google Scholar) and SsADH (Protein Data Bank accession number 1JVB) (28Esposito L. Sica F. Raia C.A. Giordano A. Rossi M. Mazzarella L. Zagari A. J. Mol. Biol. 2002; 318: 463-477Crossref PubMed Scopus (85) Google Scholar) were performed using Swisspdb viewer 3.7 (29Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9641) Google Scholar). The analysis of Ssβgly and SsADH structural topology was obtained using the TOPS (topology of protein structure) software available on the World Wide Web at www.tops.leeds.ac.uk/ (30Westhead D.R. Slidel T.W.F. Flores T.P.J. Thornton J.M. Protein Sci. 1999; 8: 897-904Crossref PubMed Scopus (106) Google Scholar). Organism and Growth—Sulfolobus solfataricus strain MT-4, isolated from acidic hot springs at Pozzuoli (Naples), was grown in vitro in a 100-liter fermenter (19Cacace M.G. De Rosa M. Gambacorta A. Biochemistry. 1976; 15: 1692-1696Crossref PubMed Scopus (10) Google Scholar), in strict accordance with the optimal conditions described for growth in vivo (i.e. at 87 °C and pH 3.0, in aerobiosis) (31De Rosa M. Gambacorta A. Nicolaus B. Buonocore V. Poerio E. Biotechnol. Lett. 1980; 2: 29-34Crossref Scopus (25) Google Scholar). The yield of bacterial biomass was about 240–280 g per 100 liters of culture broth. Amino Acid Composition Analysis—Several aliquots of homogeneous Ssβgly from different growth cycles of S. solfataricus were analyzed for their amino acid composition. The relative content of methylated lysines resulting from the analyses was between 14 and 17% (data not shown). Considering that 5 lysines of 23 (21.7%) were methylated (see below), these results indicated a constantly substoichiometric level of modification in the in vitro culture. FT-IR Spectroscopy—In the FT-IR deconvolution spectra of Ssβgly and EcSsβgly at p2H 7.0, the amide I′ bands (1700–1600-cm–1 region) of the two β-glycosidase forms were almost indistinguishable, which indicates nearly identical secondary structure contents for the two forms of the protein. Only small differences were noticed, by monitoring the amide I′ bandwidth as a function of the temperature (32Skorko-Glonek J. Lipinska B. Krzewski K. Zolese G. Bertoli E. Tanfani F. J. Biol. Chem. 1997; 272: 8974-8982Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 33Fernandez-Ballester G. Castresana J. Arrondo J.L.R. Ferragut L.A. Gonzales-Ros J.M. Biochem. J. 1992; 288: 421-426Crossref PubMed Scopus (43) Google Scholar) at p2H 7.0, in order to determine the thermal denaturation curves of the two forms of the protein (data not shown). Since this method did not appear to be sufficiently sensitive to detect subtle changes in the secondary structure, a more detailed investigation of protein thermal denaturation was performed by difference spectroscopy (32Skorko-Glonek J. Lipinska B. Krzewski K. Zolese G. Bertoli E. Tanfani F. J. Biol. Chem. 1997; 272: 8974-8982Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 34Banecki B. Zylicz M. Bertoli E. Tanfani F. J. Biol. Chem. 1992; 267: 25051-25058Abstract Full Text PDF PubMed Google Scholar, 35Tanfani F. Bertoli E. Signorini M. Bergamini C.M. Eur. J. Biochem. 1993; 218: 499-505Crossref PubMed Scopus (17) Google Scholar). Difference spectra were derived from the series of spectra recorded at stepwise increasing temperatures, by subtracting each one of them from the spectrum recorded at the next higher temperature. For example, the 95–90 °C difference spectrum corresponded to the spectrum recorded at 95 °C, after subtraction of the one recorded at 90 °C. In these spectra, a negative band indicated a lower content of a particular secondary structural element in the spectrum recorded at higher temperature and vice versa (Fig. 1, A and B). Hence, the negative broad band between 1600 and 1700 cm–1 corresponded to the loss of secondary structure in the sample recorded at higher temperature (34Banecki B. Zylicz M. Bertoli E. Tanfani F. J. Biol. Chem. 1992; 267: 25051-25058Abstract Full Text PDF PubMed Google Scholar), whereas the two positive peaks close to 1617 and 1685 cm–1 represented protein aggregation brought about by thermal denaturation (34Banecki B. Zylicz M. Bertoli E. Tanfani F. J. Biol. Chem. 1992; 267: 25051-25058Abstract Full Text PDF PubMed Google Scholar). The negative band close to 1540 cm–1 mainly represented an enhanced 1H/2H exchange, caused by the increase of temperature and by protein unfolding (36Osborne H.B. Nabedryk-Viala E. Methods Enzymol. 1982; 88: 676-680Crossref Scopus (46) Google Scholar). The difference spectra of native and recombinant proteins, shown in Fig. 1, A and B, indicated the onset of protein denaturation and aggregation. In particular, the small negative band at 1657.2 cm–1 represented a partial loss of α-helices in the samples assayed at higher temperature, whereas the positive peak close to 1617 cm–1 reflected protein aggregation. From these spectra, it appears that protein aggregation paralleled protein denaturation. The amplitudes of the negative and positive bands in the 94.2–89.0 °C difference spectrum of Ssβgly were smaller than those present in the corresponding difference spectrum of EcSsβgly. Moreover, the Tm was observed in the 99.0–98.1 °C spectrum for native Ssβgly and in the 98.1–97.2 °C spectrum for EcSsβgly (Fig. 1, A and B). When the normalized absorbance values of the broad negative band between 1600 and 1700 cm–1 and of the positive peak close to 1617 cm–1, reflecting denatu
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