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. 2008 Aug 29;283(35):23774–23781. doi: 10.1074/jbc.M802542200

FIGURE 2.

FIGURE 2.

Encapsulation of rhodanese bound to both rings of EL398 within the cavities. A, protection of encapsulated rhodanese from proteinase K. Mixtures of EL398 saturated with heat-denatured rhodanese, GroES, 200 mm glucose, and ATP were incubated for 3 s (lanes 1 and 2), 1 min (lanes 3 and 4), or 120 min (lanes 5 and 6). In the experiment denoted as ATPsingle (lanes 1 and 2), hexokinase was added to the aliquot after 3 s, and the reaction was left for 60 min longer as described for Fig. 1A. Aliquots underwent one of the two following treatments: ultrafiltration (100-kDa cutoff) and SDS-PAGE (lanes 1, 3, and 5) or ultrafiltration (100-kDa cutoff), proteinase K treatment, a second ultrafiltration (100-kDa cutoff), and SDS-PAGE (lanes 2, 4, and 6). Gels were stained with Coomassie Brilliant Blue. B, GroEL (wild-type (WT))- or EL398-assisted recovery of rhodanese activities for a single reaction cycle. A single round of folding was initiated by the addition of ATP to the mixtures containing 200 mm glucose, GroEL saturated with denatured rhodanese, and GroES. Hexokinase was then added after 3 s of the initiation. For a comparison, the ratio of the recovered activity to that at 60 min assisted by GroEL (wild-type) is shown. EL(WT), wild-type EL398.