Destabilizing mutations alter the hydrogen exchange mechanism in ribonuclease A

被引:11
作者
Bruix, Marta [1 ]
Ribo, Marc [2 ]
Benito, Antoni [2 ]
Laurents, Douglas V. [1 ]
Rico, Manuel [1 ]
Vilanova, Maria [2 ]
机构
[1] CSIC, Inst Quim Fis Rocasolano, Madrid 28006, Spain
[2] Univ Girona, Fac Ciencies, Dept Biol, Lab Enginyeria Proteines, Girona 17071, Spain
关键词
D O I
10.1529/biophysj.107.122952
中图分类号
Q6 [生物物理学];
学科分类号
071011 ;
摘要
The effect of strongly destabilizing mutations, 1106A and V108G of Ribonuclease A (RNase A), on its structure and stability has been determined by NMR. The solution structures of these variants are essentially equivalent to RNase A. The exchange rates of the most protected amide protons in RNase A (35 degrees C), the 1106A variant (35 degrees C), and the V108G variant (10 degrees C) yield stability values of 9.9, 6.0, and 6.8 kcal/mol, respectively, when analyzed assuming an EX2 exchange mechanism. Thus, the destabilization induced by these mutations is propagated throughout the protein. Simulation of RNase A hydrogen exchange indicates that the most protected protons in RNase A and the V108G variant exchange via the EX2 regime, whereas those of 1106A exchange through a mixed EX1 + EX2 process. It is striking that a single point mutation can alter the overall exchange mechanism. Thus, destabilizing mutations joins high temperatures, high pH and the presence of denaturating agents as a factor that induces EX1 exchange in proteins. The calculations also indicate a shift from the EX2 to the EX1 mechanism for less protected groups within the same protein. This should be borne in mind when interpreting exchange data as a measure of local stability in less protected regions.
引用
收藏
页码:2297 / 2305
页数:9
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