title: pH-Dependent conformational stability of the ribotoxin alpha-sarcin and four active site charge substitution variants.
abstract: Alpha-sarcin is an exquisitely specific ribonuclease that binds and cleaves a single phosphodiester bond in the large rRNA of the eukaryotic ribosome, inactivating it. To better understand this remarkable activity, the contributions of the active site residues (His 50, Glu 96, and His 137) to the conformational stability have been determined as a function of pH using variant proteins containing uncharged substitutes. Wild-type alpha-sarcin and the variants are maximally stable near pH 5.5, coinciding with the pH of optimal activity. A comparison of the stability vs pH profiles determined by thermal denaturation experiments to those calculated on the basis of pKa values shows that the charged forms of Glu 96 and His 137 compromise the enzyme's stability, lowering it. In contrast to barnase, there is little evidence for significant electrostatic interactions in the denatured states of alpha-sarcin or its active site variants between pH 3.5 and pH 8.5. Alpha-sarcin contains a long beta-hairpin and surface loops which are highly positively charged and which play key roles in membrane translocation and in ribosome binding. These positive charges decrease the stability of alpha-sarcin, particularly below pH 5. Hydrogen exchange measurements have been performed at pH 5.5 and reveal that the catalytic residues are firmly anchored in highly stable elements of secondary structure. Significant, though lower, levels of protection are observed for many amide protons in the positively charged beta-hairpin and long loops.
authors.affiliation: Departamento de Espectroscopía y Estructura Molecular, Instituto de Química-Física Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain.
authors.forename: Maria Flor
authors.initials: MF
authors.lastname: García-Mayoral
authors.forename: Alvaro Martínez
authors.initials: AM
authors.lastname: del Pozo
authors.forename: Ramón
authors.initials: R
authors.lastname: Campos-Olivas
authors.forename: José G
authors.initials: JG
authors.lastname: Gavilanes
authors.forename: Jorge
authors.initials: J
authors.lastname: Santoro
authors.forename: Manuel
authors.initials: M
authors.lastname: Rico
authors.forename: Douglas V
authors.initials: DV
authors.lastname: Laurents
authors.forename: Marta
authors.initials: M
authors.lastname: Bruix
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D004722:Endoribonucleases / Q000737:chemistry* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011489:Protein Denaturation
chemical_list: D005656:Fungal Proteins; C014801:alpha-sarcin; D004722:Endoribonucleases