Confirmation of the hierarchical folding of RNase H: a protein engineering study

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  • A Correction to this article was published on 01 May 2000

Abstract

The kinetic intermediate of RNase H is structured in a core region of the protein. To probe the role of this intermediate in the folding of RNase H, the folding kinetics of mutant proteins with altered native state stabilities were investigated. Mutations within the folding core destabilize the kinetic intermediate and slow refolding in a manner consistent with an obligatory intermediate model. Mutations outside of the folding core, however, do not affect the stability of the kinetic intermediate but do perturb the native state and transition state. These results indicate that interactions formed in the intermediate persist in the transition and native states and that RNase H folds through a hierarchical mechanism.

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Figure 1: a, Location of mutation sites in RNase H.
Figure 2: Denaturant-dependent folding kinetics of folding core mutants.
Figure 3: Denaturant-dependent kinetics of mutants outside the folding core.
Figure 4: Reaction coordinate diagrams in 0 M urea for wt* and mutant RNases H.

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Acknowledgements

We thank J. Kelly and Aviv Instrument Company for use of their stopped-flow instruments, D. King for the mass spectra, and K. Fischer, V. Pande and M. Parker for discussion and critical reading of the manuscript. This work was supported by a grant from the National Institutes of Health and a Beckman Young Investigator Award.

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Correspondence to Susan Marqusee.

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