Minimal and optimal mechanisms for GroE-mediated protein folding

We have analyzed the effects of different components of the GroE chaperonin system on protein folding by using a nonpermissive substrate (i,e., one th at has very low spontaneous refolding yield) for which rate data can be acq uired. In the absence of GroES and nucleotides, the rate of GroEL-mediated refolding of heat- and DTT-denatured mitochondrial malate dehydrogenase was extremely low, but some three times higher than the spontaneous rate. This GroEL-mediated rate was increased 17-fold by saturating concentrations of ATP, Ii-fold by ADP and GroES, and 465-fold by ATP and GroES, Optimal refol ding activity was observed when the dissociation of GroES from the chaperon in complex was dramatically reduced. Although GroEL minichaperones were abl e to bind denatured mitochondrial malate dehydrogenase, they were ineffecti ve in enhancing the refolding rate. The spectrum of mechanisms for GroE-med iated protein folding depends on the nature of the substrate, The minimal m echanism for permissive substrates (i,e,, having significant yields of spon taneous refolding), requires only binding to the apical domain of GroEL, Sl ow folding rates of nonpermissive substrates are limited by the transitions between high- and low-affinity states of GroEL alone. The optimal mechanis m, which requires holoGroEL, physiological amounts of GroES, and ATP hydrol ysis, is necessary for the chaperonin-mediated folding of nonpermissive sub strates at physiologically relevant rates under conditions in which retenti on of bound GroES prevents the premature release of aggregation-prone foldi ng intermediates from the chaperonin complex. The different mechanisms are described in terms of the structural features of mini- and hole-chaperones.