Probing the Structural Role of an αβ Loop of Maltose-binding Protein by Mutagenesis: Heat-shock Induction by Loop Variants of the Maltose-binding Protein that Form Periplasmic Inclusion Bodies

Abstract

The maltose-binding protein (MBP) of Escherichia coliis the periplasmic receptor of the maltose transport system. Previous studies have identified amino acid substitutions in an α/β loop of the structure of MBP that are critical for the in vivofolding. To probe genetically the structural role of this surface loop, we generated a library in which the corresponding codons 32 and 33 of malEwere mutagenized. The maltose phenotype, which correlates with a biologically active structure of MBP in the periplasm, indicated a considerable variability in the loop residues compatible with a correct in vivofolding pathway of the protein. By the same genetic screens, we characterized loop-variant MBPs associated with a defective periplasmic folding pathway and aggregated into inclusion bodies. Heat-shock induction with production of misfolded loop variants was examined using both lon- lacZand htrA- lacZfusions. We found that the extent of formation of inclusion bodies in the periplasm of E. coli, from misfolded loop variant MBPs, correlated with the level of heat-shock response regulated by the alternate heat-shock σ factor, σ 24.