Propagation of a single destabilizing mutation throughout the Escherichia coli ribonuclease HI native state.
A point mutation (I53A) in the core of Escherichia coli RNase H* is known to destabilize both the native conformation (DeltaG(UN)) and the kinetic intermediate (DeltaG(UI)) by 2 kcal/mole. Here, we have used native-state hydrogen deuterium exchange to ask how this destabilization is propagated throughout the molecule. Stability parameters were obtained for individual residues in I53A and compared with those from the wild-type protein. A destabilization of 2 kcal/mole was observed in residues in the core but was not detected in the periphery of the molecule. These results are consistent with the localized destabilization of the core observed in the early intermediate of the kinetic folding pathway, supporting the resemblance of this kinetic intermediate to the partially unfolded form detected in the native state at equilibrium. A thermodynamic cycle also shows no interaction between Ile 53 and a residue in the periphery. There is, however, an increase in the number of denaturant-independent exchange events in the periphery of I53A, showing that effects of the point mutation are communicated to regions outside the core, although perhaps not through changes in stability. In sum, this work shows that localized regions within a protein can be destabilized independently. Furthermore, it implies a correspondence between the kinetic intermediate and the equilibrium PUF, as the magnitude and localization of the destabilization are the same in both.
Spudich
Giulietta
G
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA.
Lorenz
Sonja
S
Marqusee
Susan
S
D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D017354:Point Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D016914:Ribonuclease H / Q000737:chemistry / Q000235:genetics*; D013816:Thermodynamics
D016914:Ribonuclease H; C078856:ribonuclease HI