Competition between DsbA-Mediated Oxidation and Conformational Folding of RTEM1 β-Lactamase

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Abstract
Similar to other proteins of the periplasm of Escherichia coli, TEM1 β-lactamase contains only a single disulfide bond. It can fold to its native conformation in both the presence and the absence of this disulfide bond. The GdmCl-dependent equilibrium unfolding of β-lactamase in vitro is well described by a N ⇌ I ⇌ U three-state model in which the native protein (N) first reacts to an intermediate of the molten globule type (I) and then to the unfolded state (U). We find that the disulfide bond of β-lactamase stabilizes I relative to U, but does not change the stability of N relative to I. The I ⇌ U transition is an extremely rapid reaction for both reduced and oxidized β-lactamase, but the N ⇌ I folding kinetics are slow and identical in the presence and the absence of the disulfide bond. This insensitivity of the N ⇌ I equilibrium and kinetics suggests that the region around the disulfide bond is already native-like folded and is presumably buried in the intermediate I, prior to the slow and rate-limiting events of folding. This was confirmed by measuring the stability of the disulfide bond, which, to a first approximation, is identical in N and I. In native, reduced β-lactamase, the thiol groups are inaccessible for oxidation by DsbA, but at the stage of the molten globule intermediate I oxidation is still possible, because I is in fast exchange with the unfolded protein U. The introduction of the disulfide bond into β-lactamase by DsbA competes with conformational folding at the stage of the final slow steps in the folding of the reduced protein. The major problem in the oxidation of proteins with one or two disulfide bonds (such as β-lactamase) is not the formation of incorrect disulfide bonds, but the premature burial of the thiol groups by the rapid conformational folding of the reduced protein. DsbA, the major thiol/disulfide isomerase of the bacterial periplasm, meets this problem. It is a very strong oxidant, and its reaction with cysteine residues in unfolded proteins is extremely fast.