Destabilizing mutations alter the hydrogen exchange mechanism in ribonuclease A

Biophysical Journal
Marta BruixMaría Vilanova

Abstract

The effect of strongly destabilizing mutations, I106A 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 I106A 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 I106A 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 ...Continue Reading

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Citations

Jun 15, 2011·Proceedings of the National Academy of Sciences of the United States of America·Rachel BernsteinSusan Marqusee
Jul 20, 2011·Biophysical Journal·Pere TubertAntoni Benito
Apr 18, 2009·Biopolymers·Roger VilàMaria Vilanova
Jul 18, 2017·Biochemistry·Pooja MalhotraJayant B Udgaonkar

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