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Folding of a mutant maltose-binding protein of Escherichia coli which forms inclusion bodies
1Département des Biotechnologies, Institut Pasteur, 25, rue du Docteur Roux, 75015 Paris, France.
Abstract:
The maltose-binding protein (MalE) of Escherichia coli is the periplasmic component of the transport system for malto-oligosaccharides. We have examined the characteristics of a Mal- mutant of malE corresponding to the double substitution Gly32 --> Asp/Ile33 --> Pro, MalE31, previously obtained by random mutagenesis. In vivo, the MalE31 precursor is efficiently processed, but the mature protein forms inclusion bodies in the periplasm. Furthermore, the accumulation of insoluble MalE31 is independent of its cellular localization; MalE31 lacking its signal sequence forms inclusion bodies in the cytoplasm. The native MalE31 protein can be purified by affinity chromatography from inclusion bodies after denaturation by 8 M urea. The renatured protein exhibits full maltose binding affinity (Kd= 9 x 10(-7) M), suggesting that its folded structure is similar to that of the wild-type protein. Unfolding/refolding experiments show that MalE31 is less stable (-5. 5 kcal/mol) than the wild-type protein (-9.5 kcal/mol) and that folding intermediates have a high tendency to form aggregates. In conclusion, the observed phenotype of cells expressing malE31 can be explained by a defective folding pathway of the protein.
Insights
A mutant maltose-binding protein (MalE31) in Escherichia coli forms insoluble inclusion bodies due to a defective folding pathway. Despite aggregation, the renatured MalE31 retains maltose-binding affinity, indicating structural similarities to the wild-type protein.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Folding
Background:
- The maltose-binding protein (MalE) is crucial for malto-oligosaccharide transport in Escherichia coli.
- A specific mutant, MalE31 (Gly32Asp/Ile33Pro), was previously generated via random mutagenesis.
Purpose of the Study:
- To characterize the in vivo and in vitro properties of the MalE31 mutant protein.
- To elucidate the molecular basis for the observed cellular phenotype associated with MalE31 expression.
Main Methods:
- In vivo expression and cellular localization studies.
- Protein purification using affinity chromatography under denaturing conditions.
- In vitro unfolding/refolding experiments and binding affinity measurements.
Main Results:
- MalE31 precursor is efficiently processed, but mature MalE31 forms insoluble inclusion bodies in both periplasm and cytoplasm.
- Renatured MalE31 exhibits wild-type maltose binding affinity (Kd= 9 x 10(-7) M).
- MalE31 is thermodynamically less stable than wild-type MalE, with folding intermediates prone to aggregation.
Conclusions:
- The MalE31 mutation leads to a defective protein folding pathway, resulting in aggregation and inclusion body formation.
- The aggregation is independent of cellular localization.
- Despite aggregation, the core maltose-binding function is preserved upon refolding.