Inactive GroEL monomers can be isolated and reassembled to functional tetradecamers that contain few bound peptides.

For the first time, it has been shown that GroEL can be converted from tetradecamers (14-mers) to monomers under conditions commonly used for the preparation of this chaperonin. The essential requirements are the simultaneous presence of nucleotides such as MgATP or MgADP and a solid-phase anion-exchange medium. The monomers that are formed are metastable in that they only reassemble to a small degree in the absence of additives. These results are in keeping with previous studies on high pressure dissociation that showed the separated monomers display conformational plasticity and can undergo conformational relaxation when relieved of the constraints of the quaternary structure in the oligomer (Gorovits, B., Raman, C. S., and Horowitz, P. M. (1995) J. Biol. Chem. 270, 2061-2066). The monomers display greatly enhanced hydrophobic exposure to the probe 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid, although they are not active in folding functions, and they are unable to form complexes with partially folded rhodanese. The monomers can be completely reassembled to 14-mers by incubation in 1 M ammonium sulfate. There is no evidence of intermediates in the reassembly process. Compared with the original oligomers, the reassembled 14-mers have (a) very low levels of polypeptide contaminants and tryptophan-like fluorescence, two problems that previously hampered spectroscopic studies of GroEL structure and function; (b) functional properties that are very similar to the original material; (c) considerably decreased hydrophobic exposure in the native state; and (d) a similar triggered exposure of hydrophobic surfaces after treatment with urea or spermidine. This study demonstrates that the quaternary structure of GroEL is more labile than previously thought. These results are consistent with suggestions that nucleotides can loosen subunit interactions and show that changes in quaternary structure can operate under conditions where GroEL function has been demonstrated.