Buried, Charged, Non-Ion-Paired Aspartic Acid 76 Contributes Favorably to the Conformational Stability of Ribonuclease T1

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• 作者：Anthony Giletto and C. Nick Pace
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：October 5, 1999
• 年：1999
• 卷：38
• 期：40
• 页码：13379 - 13384
• 全文大小：90K
• 年卷期：v.38,no.40(October 5, 1999)
• ISSN：1520-4995

文摘

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 Å) and a 2.66 Åhydrogen 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 stabilitywas measured as a function of pH for wild-type RNase T1 and the D76N mutant and showed that the pHdependence below pH 3 was almost entirely due to Asp 76. The pK of Asp 76 is 0.5 in the native stateand 3.7 in the denatured state. Thus, the hydrogen bonding of the carboxyl group of Asp 76 contributesmore than half of the net stability of RNase T1 at pH 7. In addition, the charged carboxyl of Asp 76stabilizes structure in the denatured states of RNase T1 that is not present in D76N, D76S, and D76A.