Folding of a mutant maltose-binding protein of Escherichia coli which forms inclusion bodies.
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.
Betton
J M
JM
Département des Biotechnologies, Institut Pasteur, 25, rue du Docteur Roux, 75015 Paris, France.
Hofnung
M
M
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins / Q000737:chemistry*; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry*; D002451:Cell Compartmentation; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012995:Solubility
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C461599:maltose transport system, E coli