PROBING THE STRUCTURAL ROLE OF AN ALPHA-BETA LOOP OF MALTOSE-BINDING PROTEIN BY MUTAGENESIS - HEAT-SHOCK INDUCTION BY LOOP VARIANTS OF THE MALTOSE-BINDING PROTEIN THAT FORM PERIPLASMIC INCLUSION-BODIES

The maltose-binding protein (MBP) of Escherichia coli is the periplasm ic receptor of the maltose transport system. Previous studies have ide ntified amino acid substitutions in an alpha/beta loop of the structur e of MBP that are critical for the in vivo folding. To probe genetical ly the structural role of this surface loop, we generated a library in which the corresponding codons 32 and 33 of malE were mutagenized. Th e maltose phenotype, which correlates with a biologically active struc ture of MBP in the periplasm, indicated a considerable variability in the loop residues compatible with a correct in vivo folding pathway of the protein. By the same genetic screens, we characterized loop-varia nt MBPs associated with a defective periplasmic folding pathway and ag gregated into inclusion bodies. Heat-shock induction with production o f misfolded loop variants was examined using both lon-lacZ and htrA-la cZ fusions. We found that the extent of formation of inclusion bodies in the periplasm of E. coli, from misfolded loop variant MBPs, correla ted with the level of heat-shock response regulated by the alternate h eat-shock sigma factor, sigma(24). (C) 1996 Academic Press Limited