The chaperonin GroEL can assist protein folding and normally acts with the co-chaperonin GroES. These Escherichia coli proteins are encoded on the same operon, with GroES positioned first. In this report, we have investigated the reversible folding of GroES. Using fluorescence anisotropy of dansyl-labeled GroES, intrinsic fluorescence, bis-ANS binding, sedimentation velocity, and limited proteolysis, we show that GroES unfolds in a single, two state transition. Importantly, intrinsic fluorescence and sedimentation velocity analyses show that GroES is capable of refolding and reassembling from a urea denatured state. The refolded GroES is fully active as shown by its ability to assist GroEL in the refolding of rhodanese. These results indicate that chaperonins may not require other chaperonins for successful folding/assembly. We also show that GroES is capable of assisting in the refolding/reassembly of fully denatured GroEL. The reversible folding of GroES coupled with the ability of GroES to assist the refolding/reassembly of GroEL suggest that the groE operon may be organized in a manner that provides a structural role in GroES/GroEL assembly as well as a functional role.
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