title: Thermal stability and aggregation of sulfolobus solfataricus beta-glycosidase are dependent upon the N-epsilon-methylation of specific lysyl residues: critical role of in vivo post-translational modifications.
abstract: 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 beta-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 beta-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 beta-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 beta-glycosidase from S. solfataricus.
authors.affiliation: Institute of Protein Biochemistry, CNR, Napoli, Italy. f.febbraio@ibp.cnr.it
authors.forename: Ferdinando
authors.initials: F
authors.lastname: Febbraio
authors.forename: Annapaola
authors.initials: A
authors.lastname: Andolfo
authors.forename: Fabio
authors.initials: F
authors.lastname: Tanfani
authors.forename: Raffaella
authors.initials: R
authors.lastname: Briante
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Gentile
authors.forename: Silvestro
authors.initials: S
authors.lastname: Formisano
authors.forename: Carlo
authors.initials: C
authors.lastname: Vaccaro
authors.forename: Andrea
authors.initials: A
authors.lastname: Scirè
authors.forename: Enrico
authors.initials: E
authors.lastname: Bertoli
authors.forename: Piero
authors.initials: P
authors.lastname: Pucci
authors.forename: Roberto
authors.initials: R
authors.lastname: Nucci
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D004396:Coloring Agents / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005959:Glucosidases / Q000737:chemistry*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008239:Lysine / Q000737:chemistry*; D013058:Mass Spectrometry; D008745:Methylation; D008956:Models, Chemical; D008958:Models, Molecular; D010078:Oxazines / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D012984:Software; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013055:Spectrophotometry, Infrared; D017550:Spectroscopy, Fourier Transform Infrared; D016944:Sulfolobus / Q000201:enzymology*; D013696:Temperature
chemical_list: D000596:Amino Acids; D004396:Coloring Agents; D003902:Detergents; D010078:Oxazines; D011994:Recombinant Proteins; D005959:Glucosidases; C104851:Sulfolobus solfataricus beta-glycosidase; D008239:Lysine; C044808:nile red