Stabilization by GroEL, a Molecular Chaperone, and a Periplasmic Fraction, as Well as Refolding in the Presence of Dithiothreitol, of Acid-Unfolded Dimethyl Sulfoxide Reductase, a Periplasmic Protein of Rhodobacter sphaeroides f. sp. denitrificans

Abstract

The mechanisms of folding of a periplasmic protein was studied in vitro using dimethyl sulfoxide reductase (DMSOR), a periplasmic enzyme of Rhodobacter sphaeroides f. sp. denitrificans. When DMSOR was denatured by acidification to pH 2 at 30°C, the molybdenum cofactor was immediately released and unfolded forms of DMSOR appeared within 2 min. When the acid-unfolded DMSOR has been incubated in refolding buffer (pH 8.0) at 20°C for 2 h, it became almost undetectable after electrophoresis on a non-denaturing gel. This result suggests that the acid-unfolded DMSOR might have aggregated after incubation. The aggregation was suppressed by incubation in the presence of commercial GroEL, a molecular chaperone. When reduced dithiothreitol (DTT) was added to the acid-unfolded forms in the presence of GroEL, some of the DMSOR was converted to the native form, which had the same mobility on a non-denaturing gel as the active emzyme. Non-reducing SDS-polyacrylamide gel electrophoresis of the acid-unfolded forms of DMSOR indicated that the unfolded forms were a mixture of heterogeneous ly folded or misfolded forms and that their forms were converted by DTT to the fully reduced form. The periplasmic fraction of the phototroph was also able to suppress the aggregation of the acid-unfolded DMSOR, and a protein(s) with a molecular mass of about 40 kDa in the periplasm was revealed to have stabilizing activity. It appears that there exists a mechanism whereby the unfolded DMSOR that is secreted into the periplasm is maintained in a non-aggregated and reduced form during folding to the native form.