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The time course of refolding of both pig muscle and yeast 3-phosphoglycerate kinase (molecular masses about 47 kDa), as well as their proteolytic C-terminal fragments (30 and 33 kDa, respectively) has been investigated. Very similar refolding kinetics (with half-time between 80-120 s, at 20°C) were observed by fluorescence and ultraviolet absorbance spectroscopy, as well as by activity measurements, for the intact enzyme from both sources. This time course appears not to depend on the time the protein spends in the unfolded state, i.e. it is certainly not controlled by proline isomerization. Furthermore, after removal of a large N-terminal part (molecular mass of about 18 kDa for pig muscle enzyme or 13 kDa for yeast enzyme) of the molecule by proteolysis, refolding of the remaining C-terminal fragment of both proteins follows kinetics virtually indistinguishable from those of the intact protein molecule.The refolding of simple globular proteins occurs on a wide range of time scale, from the subsecond region [l, 21 up to minutes or hours [2-61. In some cases this process can be resolved into kinetically distinguishable steps (e. g. [7, 81). However, the nature of the folding intermediates and the ratedetermining steps could rarely be identified (cf. reviews [9, 101). If the protein contains no disulfide bridges, the only reason known for slow refolding is the isomerization around the proline imide peptide bond [l 1 -151 which can be accelerated by a specific enzyme, proline cis-trans-isomerase [13, 151. Apart from this, other types of unknown slow conformational processes might also be operative in the folding pathway. For example, kinetic investigation of horse muscle 3-phosphoglycerate kinase refolding revealed a slow phase which was not attributable to proline isomerization [16]. This fact requires special attention, since it may reflect the presence of an unknown rate-limiting factor in protein refolding. It is possible that the slow refolding of 3-phosphoglycerate kinase is somehow associated with the two-domain structural nature of the molecule as the correct domain assembly can be the rate-limiting step. A useful approach to clarify this point is the separation of the C-terminal and N-terminal parts of the molecule and the study of their refolding separately [24]. Various efforts have succeeded in producing large fragments (or domains) of the enzyme molecule either by analytical 1251 and proteolytic [26, 271 cleavage or by genetic engineering [28, 291. Renaturation experiments with the fragments supported their ability to fold independently both for horse muscle [26] and yeast [25, 301 3-phosphoglycerate kinases. Nevertheless, no kinetic experiment has yet been performed to study the refolding of the isolated fragments of this enzyme. We first showed the possibility of reactivation of pig muscle 3-phosphoglycerate kinase from its isolated and unfolded proteolytic fragments [31]. To understand further the rate-limiting step of the protein refolding, we investigated the refolding of one isolated C-termin...

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Molecular chaperones are a special class of heat shock proteins (Hsp) that assist the folding and formation of the quaternary structure of other proteins both in vivo and in vitro. However, some chaperones are complex oligomeric proteins, and one of the intriguing questions is how the chaperones fold. The representatives of the Escherichia coli chaperone system GroEL (Hsp60) and GroES (Hsp10) have been studied most intensively. GroEL consists of 14 identical subunits combined into two interacting ring-like structures of seven subunits each, while the co-chaperone GroES interacting with GroEL consists of seven identical subunits combined into a dome-like oligomeric structure. In spite of their complex quaternary structure, GroEL and GroES fold well both in vivo and in vitro. However, the specific oligomerization of GroEL subunits is dependent on ligands and external conditions. This review analyzes the literature and our own data on the study of unfolding (denaturation) and refolding (renaturation) processes of these molecular chaperones and the effect of ligands and solvent composition. Such analysis seems to be useful for understanding the folding mechanism not only of the GroEL/GroES complex, but also of other oligomeric protein complexes.

Escherichia coli heat-shock proteins GroEL and GroES stimulate (in an ATP-dependent manner) the folding of various proteins. In this study scanning microcalorimetry was applied to investigate GroEL thermostability in the presence of its ligands. Mg 2+ and K + ions stabilize while ADP destabilizes the GroEL molecule against the action of temperature. Furthermore, ADP essentially increases the number of binding sites for the hydrophobic probe (ANS) and the number of GroEL SH-groups accessible to Ellman's reagent as well as the accessibility of the protein to the action of trypsin. The interaction of GroEL with GroES in the presence of Mg 2+ -ADP eliminates the destabilizing effect of ADP on the GroEL molecule against the action of temperature and Ellman's reagent but does not change its hydrophobicity and accessibility to trypsin.