Abstract
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.
Highlights
Aggregation that competes with the acquisition of native structure [1, 2]
These results provide a system with distinct advantages for studying some of the details of GroEL function
3) The reassembled 14-mers contain very low levels of peptide contaminants and tryptophan fluorescence, which have confounded previous studies of GroEL. 4) The subunit interactions are substantially altered by conditions required for GroEL function
Summary
The most efficient release of properly folded target proteins from GroEL requires the use of the co-chaperonin GroES (cpn10) and MgATP, it has been demonstrated that folding can be influenced by GroEL alone [3]. The individual monomers within the oligomer are folded into three domains: equatorial, intermediate, and apical. It has been suggested that quaternary structural changes in the 14-mer are important for binding proteins and protecting hydrophobic surfaces from nonproductive interactions as well as for transmitting information for cooperative binding and release in processes involving GroES interactions [8]. We report that it is possible to prepare monomeric forms of GroEL under native conditions. The formation of GroEL monomers is easier than formerly suspected, and previous preparative procedures produced monomeric GroEL during intermediate stages [13]. It is possible to produce, in a way related to function, GroEL with little detectable tryptophan fluorescence and barely detectable levels of
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