Destabilizing mutations alter the hydrogen exchange mechanism in ribonuclease A.
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 local stability in less protected regions.
Bruix
Marta
M
Instituto de Química Física "Rocasolano", Consejo Superior de Investigaciones Científicas, 28006 Madrid, Spain. mbruix@iqfr.csic.es
Ribó
Marc
M
Benito
Antoni
A
Laurents
Douglas V
DV
Rico
Manuel
M
Vilanova
Maria
M
D001703:Biophysics / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D006859:Hydrogen / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D011522:Protons; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000235:genetics*; D013696:Temperature; D013816:Thermodynamics
D011522:Protons; D006859:Hydrogen; D012259:Ribonuclease, Pancreatic