pH-Dependent Conformational Stability of the Ribotoxin -Sarcin and Four Active Site Charge Substitution Variants
详细信息 查看全文
- 作者：Maria Flor Garcí ; a-Mayoral ; Á ; lvaro Martí ; nez del Pozo ; Ramó ; n Campos-Olivas ; José ; G. Gavilanes ; Jorge Santoro ; Manuel Rico ; Douglas V. Laurents ; Marta Bruix
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：November 21, 2006
- 年：2006
- 卷：45
- 期：46
- 页码：13705 - 13718
- 全文大小：475K
- 年卷期：v.45,no.46(November 21, 2006)
- ISSN：1520-4995
文摘
-Sarcin is an exquisitely specific ribonuclease that binds and cleaves a single phosphodiesterbond in the large rRNA of the eukaryotic ribosome, inactivating it. To better understand this remarkableactivity, the contributions of the active site residues (His 50, Glu 96, and His 137) to the conformationalstability have been determined as a function of pH using variant proteins containing uncharged substitutes.Wild-type -sarcin and the variants are maximally stable near pH 5.5, coinciding with the pH of optimalactivity. A comparison of the stability vs pH profiles determined by thermal denaturation experiments tothose calculated on the basis of pKa values shows that the charged forms of Glu 96 and His 137 compromisethe enzyme's stability, lowering it. In contrast to barnase, there is little evidence for significant electrostaticinteractions in the denatured states of -sarcin or its active site variants between pH 3.5 and pH 8.5.-Sarcin contains a long 
-hairpin and surface loops which are highly positively charged and which playkey roles in membrane translocation and in ribosome binding. These positive charges decrease the stabilityof -sarcin, particularly below pH 5. Hydrogen exchange measurements have been performed at pH 5.5and 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 positivelycharged -hairpin and long loops.