TERTIARY STRUCTURE-DEPENDENCE OF MISFOLDING SUBSTITUTIONS IN LOOPS OFTHE MALTOSE-BINDING PROTEIN

We previously identified and characterized amino acid substitutions in a loop connecting helix I to strand B, the alpha I/beta B loop, of th e N-domain that are critical for in vivo folding of the maltose-bindin g protein (MalE31). The tertiary context-dependence of this mutation i n MalE folding was assessed by probing the tolerance of an equivalent alpha beta loop of the C-domain to the same amino acid substitutions ( MalE219). Moving the loop mutation from the N- to the C-domain elimina ted the in vivo misfolding step that led to the formation of inclusion bodies. In vitro, both loop variants exhibited an important decrease of stability, but their intrinsic tendency to aggregate was well corre lated with their periplasmic fates in Escherichia coli Furthermore, th e noncoincidence of the unfolding and refolding transition curves and increase of light scattering during the refolding of MalE31 indicate t hat a competing off-pathway reaction could occurs on the folding pathw ay of this variant. These results strongly support the notion that the formation of supersecondary structures of the N-domain is a rate-limi ting step in the folding pathway of MalE.