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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°C), the I106A variant (35°C), and the V108G variant (10°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.
INTRODUCTION
The ability of enzymes to carry out molecular jobs with high efficacy and specificity make them good candidates for a variety of applications including medicine, biotechnology, or nanotechnology. Most of these applications require protein modifications to improve some fundamental characteristics. like stability, with respect to the wild-type protein. Ribonuclease A (RNasc A) (Fig. 1) is one of the most and best studied enzymes from the structural, enzymatic, folding and stability perspectives (1,2). Recently some designed variants and natural homologs of RNase A have found important applications in biomedicine. On the other hand, point mutations strongly destabilizing the native structure of proteins can lead to loss of function or to the formation of non-native and toxic amyloid conformations, which have been implicated in over 20 mortal human diseases (3). In RNase A. residues I106 and V108 are buried deep in the hydrophobic core and are thought to contribute to the early folding of the protein. The contribution of these residues to the stability and folding of RNase A has been probed by...