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 Gly Asp/Ile 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 (K= 9 10M), 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.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				M. Masi, G. Duret, A. H. Delcour, and R. Misra Folding and trimerization of signal sequence-less mature TolC in the cytoplasm of Escherichia coli Microbiology, June 1, 2009; 155(6): 1847 - 1857. [Abstract] [Full Text] [PDF]

				N. Rutherford, M.-E. Charbonneau, F. Berthiaume, J.-M. Betton, and M. Mourez The Periplasmic Folding of a Cysteineless Autotransporter Passenger Domain Interferes with Its Outer Membrane Translocation J. Bacteriol., June 1, 2006; 188(11): 4111 - 4116. [Abstract] [Full Text] [PDF]

				I. L. Grigorova, R. Chaba, H. J. Zhong, B. M. Alba, V. Rhodius, C. Herman, and C. A. Gross Fine-tuning of the Escherichia coli {sigma}E envelope stress response relies on multiple mechanisms to inhibit signal-independent proteolysis of the transmembrane anti-sigma factor, RseA Genes & Dev., November 1, 2004; 18(21): 2686 - 2697. [Abstract] [Full Text] [PDF]

				S. S. Mansy, S.-p. Wu, and J. A. Cowan Iron-Sulfur Cluster Biosynthesis: BIOCHEMICAL CHARACTERIZATION OF THE CONFORMATIONAL DYNAMICS OF THERMOTOGA MARITIMA IscU AND THE RELEVANCE FOR CELLULAR CLUSTER ASSEMBLY J. Biol. Chem., March 12, 2004; 279(11): 10469 - 10475. [Abstract] [Full Text] [PDF]

				B. Collinet, H. Yuzawa, T. Chen, C. Herrera, and D. Missiakas RseB Binding to the Periplasmic Domain of RseA Modulates the RseA:sigma E Interaction in the Cytoplasm and the Availability of sigma E{middle dot}RNA Polymerase J. Biol. Chem., October 20, 2000; 275(43): 33898 - 33904. [Abstract] [Full Text] [PDF]

				M. K. B. Berlyn Linkage Map of Escherichia coli K-12, Edition 10: The Traditional Map Microbiol. Mol. Biol. Rev., September 1, 1998; 62(3): 814 - 984. [Abstract] [Full Text] [PDF]

				J.-M. Betton, N. Sassoon, M. Hofnung, and M. Laurent Degradation versus Aggregation of Misfolded Maltose-binding Protein in the Periplasm of Escherichia coli J. Biol. Chem., April 10, 1998; 273(15): 8897 - 8902. [Abstract] [Full Text] [PDF]

				K. Uhland, M. Mondigler, C. Spiess, W. Prinz, and M. Ehrmann Determinants of Translocation and Folding of TreF, a Trehalase of Escherichia coli J. Biol. Chem., July 28, 2000; 275(31): 23439 - 23445. [Abstract] [Full Text] [PDF]

				X.-q. Liu and C.-C. Wang Disulfide-dependent Folding and Export of Escherichia coli DsbC J. Biol. Chem., January 5, 2001; 276(2): 1146 - 1151. [Abstract] [Full Text] [PDF]