FOLDING OF A MUTANT MALTOSE-BINDING PROTEIN OF ESCHERICHIA-COLI WHICHFORMS INCLUSION-BODIES

The maltose-binding protein (MalE) of Escherichia coli is the periplas mic component of the transport system for malto-oligosaccharides. We h ave examined the characteristics of a Mal(-) mutant of malE correspond ing to the double substitution Gly(32) --> Asp/Ile(33) --> Pro, MalE31 , previously obtained by random mutagenesis. In vivo, the MalE31 precu rsor is efficiently processed, but the mature protein forms inclusion bodies in the periplasm. Furthermore, the accumulation of insoluble Ma lE31 is independent of its cellular localization; MalE31 lacking its s ignal sequence forms inclusion bodies in the cytoplasm, The native Mal E31 protein can be purified by affinity chromatography from inclusion bodies after denaturation by 8 M urea, The renatured protein exhibits full maltose binding affinity (K-d = 9 x 10(-7) M), suggesting that it s folded structure is similar to that of the wild type protein. Unfold ing/refolding experiments show that MalE31 is less stable (-5.5 kcal/m ol) than the wild-type protein (-9.5 kcal/mol) and that folding interm ediates have a high tendency to form aggregates, In conclusion, the ob served phenotype of cells expressing malE31 can be explained by a defe ctive folding pathway of the protein.