Article ID Journal Published Year Pages File Type
2189387 Journal of Molecular Biology 2006 11 Pages PDF
Abstract

The ionizable groups in proteins with the lowest pKs are the carboxyl groups of aspartic acid side-chains. One of the lowest, pK = 0.6, is observed for Asp76 in ribonuclease T1. This low pK appeared to result from hydrogen bonds to a water molecule and to the side-chains of Asn9, Tyr11, and Thr91. The results here confirm this by showing that the pK of Asp76 increases to 1.7 in N9A, to 4.0 in Y11F, to 4.2 in T91V, to 4.4 in N9A + Y11F, to 4.9 in N9A + T91V, to 5.9 in Y11F + T91V, and to 6.4 in the triple mutant: N9A + Y11F + T91V. In ribonuclease Sa, the lowest pK = 2.4 for Asp33. This pK increases to 3.9 in T56A, which removes the hydrogen bond to Asp33, and to 4.4 in T56V, which removes the hydrogen bond and replaces the –OH group with a –CH3 group. It is clear that hydrogen bonds are able to markedly lower the pK values of carboxyl groups in proteins. These same hydrogen bonds make large contributions to the conformational stability of the proteins. At pH 7, the stability of D76A ribonuclease T1 is 3.8 kcal mol−1 less than wild-type, and the stability of D33A ribonuclease Sa is 4.1 kcal mol−1 less than wild-type. There is a good correlation between the changes in the pK values and the changes in stability. The results suggest that the pK values for these buried carboxyl groups would be greater than 8 in the absence of hydrogen bonds, and that the hydrogen bonds and other interactions of the carboxyl groups contribute over 8 kcal mol−1 to the stability.

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Life Sciences Biochemistry, Genetics and Molecular Biology Cell Biology
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