Buried, charged, non-ion-paired aspartic acid 76 contributes favorably to the conformational stability of ribonuclease T1

被引:70
作者
Giletto, A
Pace, CN [1 ]
机构
[1] Texas A&M Univ, Dept Med Biochem & Genet, College Stn, TX 77843 USA
[2] Texas A&M Univ, Dept Biochem & Biophys, College Stn, TX 77843 USA
[3] Texas A&M Univ, Ctr Macromol Design, College Stn, TX 77843 USA
关键词
D O I
10.1021/bi991422s
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The side-chain carboxyl of Asp 76 in ribonuclease: T1 (RNase T1) is buried, charged, non-ion-paired, and forms three good intramolecular hydrogen bonds (2.63, 2.69, and 2.89 Angstrom) and a 2.66 Angstrom hydrogen bond to a buried, conserved water molecule. When Asp 76 was replaced by Asn, Ser, and Ala, the conformational stability of the protein decreased by 3.1, 3.2, and 3.7 kcal/mol, respectively. The stability was measured as a function of pH for wild-type RNase T1 and the D76N mutant and showed that the pH dependence below pH 3 was almost entirely due to Asp 76. The pK of Asp 76 is 0.5 in the native state and 3.7 in the denatured state. Thus, the hydrogen bonding of the carboxyl group of Asp 76 contributes more than half of the net stability of RNase T1 at pH 7. In addition, the charged carboxyl of Asp 76 stabilizes structure in the denatured states of RNase TI that is not present in D76N, D76S, and D76A.
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页码:13379 / 13384
页数:6
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