pH-Dependent Conformational Stability of the Ribotoxin α-Sarcin and Four Active Site Charge Substitution Variants^†

α-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 α-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 pK_a 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 α-sarcin or its active site variants between pH 3.5 and pH 8.5. α-Sarcin contains a long β-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 α-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 β-hairpin and long loops.