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Propagation of a single destabilizing mutation throughout the Escherichia coli ribonuclease HI native state.

ABSTRACT: 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.

SUBMITTER: Spudich G

PROVIDER: S-EPMC2373462 | biostudies-literature | 2002 Mar

REPOSITORIES: biostudies-literature

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Propagation of a single destabilizing mutation throughout the Escherichia coli ribonuclease HI native state.

Spudich Giulietta G Lorenz Sonja S Marqusee Susan S

Protein science : a publication of the Protein Society 20020301 3

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 ...[more]

PMID: 11847275

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Project description:BACKGROUND: Eukaryotic Ribonuclease inhibitor (RI), belonging to the RNH1 family, is distinguished by unique features--a high sensitivity to oxidation due to the large number of reduced cysteins and a high hydrophobicity, which made most production approaches so far unsuccessful or resulted in very low yields. In this work efficient in vivo folding of native RI in the Escherichia coli cytoplasm was obtained by external addition of a reducing agent in tandem with oxygen limitation and overproduction of a molecular chaperonin. After optimisation of the production conditions in the shake flask scale the process was scaled up to high cell densities by applying a glucose limited fed-batch procedure. RESULTS: RI production in a T7 RNA polymerase based system results in accumulation of aggregated inactive product in inclusion bodies. Combination of addition of the reductant DTT, low production temperature and coexpression of the chaperonin GroELS resulted in high level production of approximately 25 mg g(-1) CDW active RI in E. coli ER2566 pET21b, corresponding to approximately 800 kU g(-1) cell wet weight. Further conditional screening under fed-batch-like conditions with the EnBase® technology and scale up into the bioreactor scale resulted in an efficient high cell density glucose and oxygen limited fed-batch process with a final cell dry weight of 25 g L(-1) and a total RI yield of app. 625 mg L(-1) (volumetric activity of 80,000 kU L(-1)). The E. coli based production constructs showed a very high robustness. The recombinant culture maintained its productivity despite the combination of the toxic growth conditions, the substrate limited production mode in tandem with a high level expression of several recombinant proteins, the set of molecular chaperonins and the target protein (RI). CONCLUSIONS: High level production of active RI in E. coli in a T7 RNA polymerase expression system depends on the following factors: (i) addition of a reducing agent, (ii) low production temperature, (iii) oxygen limitation, and (iii) co-overexpression of the chaperonin GroELS. The study indicates the strength of applying fed-batch cultivation techniques for the efficient optimisation of production factors already at the screening stage for fast and straight forward bioprocess development even for target proteins which show a complex folding behaviour. In our case none of the approaches alone would have resulted in significant accumulation of active RI.

Dataset Information

Propagation of a single destabilizing mutation throughout the Escherichia coli ribonuclease HI native state.

Publications

Propagation of a single destabilizing mutation throughout the Escherichia coli ribonuclease HI native state.

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