GroEL-assisted and -unassisted refolding of mature and precursor adrenodoxin: the role of the precursor sequence.

We have performed refolding studies on a [2Fe-2S] protein, adrenodoxin (Adx), and its precursor form, preadrenodoxin. In vitro, mature Adx is expressed as a soluble active form in Escherichia coli, but precursor Adx is expressed in inclusion bodies. Both mature and precursor Adx refolded spontaneously from their denatured forms and the recovery levels of enzyme activities were 40 and 37% for mature and precursor Adx, respectively. Furthermore, the interaction between GroEL- and Gdn-HCl-denatured mature and precursor forms was investigated. In the case of mature Adx, the activity was increased in the presence of either GroEL, GroES, or bovine serum albumin and the refolding of mature Adx is a nonspecific process. However, the GroEL-mediated reaction is specific for precursor Adx under the experimental conditions used here. A higher electron transfer activity is obtained after ATP addition to the GroEL-containing refolding mixture, and GroEL-precursor complexes were found by gel chromatography studies. Our observation suggests that the small single-domain protein Adx (mature form) folded independently of the chaperonin GroEL. The contribution of the chaperonin complexes to the folding is toward the aggregation-sensitive precursor Adx, which in vitro folded 1.3- to 1.4-fold slower than mature Adx. This demonstrates that the presequence is responsible for the formation of inclusion bodies and for the in vitro recognition motif for GroEL binding.