Chaperonin-affected folding of globular proteins.

We studied the effect of GroEL on the kinetic refolding ofα-lactalbumin by stopped-flow fluorescence techniques. We usedwild-type GroEL and its ATPase-defficient mutant D398A, and studied thebinding constants between GroEL and the molten globule foldingintermediate at various concentrations of ADP and ATP. The results arecompared with titration of GroEL with the nucleotides, ADP, ATP-analogs(ATP-γS and AMP-PNP) and ATP, which have shown that bothADP and the ATP analogs are bound to GroEL in a non-cooperativemanner but that ATP shows a cooperative effect. Similarly, the bindingconstant between GroEL and the folding intermediate decreased in acooperative manner with an increase in ATP concentration although itshowed non-cooperative decrease with respect to ADP concentration. Itis shown that the allosteric control of GroEL by the nucleotides isresponsible for the above behavior of GroEL and that the observeddifference between the ATP- and ADP-induced transitions of GroEL isbrought about by a small difference in an allosteric parameter (the ratio ofthe nucleotide affinities of GroEL in the high-affinity and the low-affinitystates), i.e., 4.1 for ATP and 2.6 for ADP.

Nucleotide-induced transition of GroEL from the high-affinity to the low-affinity state for a target protein: effects of ATP and ADP on the GroEL-affected refolding of alpha-lactalbumin.

We studied the refolding kinetics of alpha-lactalbumin in the presence of wild-type GroEL and its ATPase-deficient mutant D398A at various concentrations of nucleotides (ATP and ADP). We evaluated the apparent binding constant between GroEL and the alpha-lactalbumin refolding intermediate quantitatively by numerical simulation analysis of the alpha-lactalbumin refolding curves in the presence and absence of GroEL. The binding constant showed a co-operative decrease with an increase in ATP concentration, whereas the binding constant decreased in a non-co-operative manner with respect to ADP concentration. For the D398A mutant, the ATP-induced decrease in affinity occurred much faster than the steady-state ATP hydrolysis by this mutant, suggesting that ATP binding to GroEL rather than ATP hydrolysis, was responsible for the co-operative decrease in the affinity for the target protein. We thus analyzed the nucleotide-concentration dependence of affinity of GroEL for the target protein using an allosteric Monod-Wyman-Changeux model in which GroEL underwent an ATP-induced co-operative conformational transition between the high-affinity and low-affinity states of the target protein. The transition midpoint of the ATP-induced transition of GroEL has been found to be around 30 microM, in good agreement with the midpoint evaluated in other structural studies of GroEL. The results show that the observed difference between ATP and ADP-induced transitions of GroEL are brought about by a small difference in an allosteric parameter (the ratio of the nucleotide affinities of GroEL in the high-affinity and the low-affinity states), i.e. 4.1 for ATP and 2.6 for ADP.

Nucleotide binding to the chaperonin GroEL: non-cooperative binding of ATP analogs and ADP, and cooperative effect of ATP.

Chaperonin-assisted protein folding proceeds through cycles of ATP binding and hydrolysis by GroEL, which undergoes a large structural change by the ATP binding or hydrolysis. One of the main concerns of GroEL is the mechanism of the productive and cooperative structural change of GroEL induced by the nucleotide. We studied the cooperative nature of GroEL by nucleotide titration using isothermal titration calorimetry and fluorescence spectroscopy. Our results indicated that the binding of ADP and ATP analogs to a single ring mutant (SR1), as well as that to GroEL, was non-cooperative. Only ATP induces an apparently cooperative conformational change in both proteins. Furthermore, the fluorescence changes of pyrene-labeled GroEL indicated that GroEL has two kinds of nucleotide binding sites. The fluorescence titration result fits well with a model in which two kinds of binding sites are both non-cooperative and independent of each other. These results suggest that the binding and hydrolysis of ATP may be necessary for the cooperative transition of GroEL.

Hydrophilic residues at the apical domain of GroEL contribute to GroES binding but attenuate polypeptide binding.

The GroES binding site at the apical domain of GroEL, mostly consisting of hydrophobic residues, overlaps largely with the substrate polypeptide binding site. Essential contribution of hydrophobic interaction to the binding of both GroES and polypeptide was exemplified by the mutant GroEL(L237Q) which lost the ability to bind either of them. The binding site, however, contains three hydrophilic residues, E238, T261, and N265. For GroES binding, N265 is essential since GroEL(N265A) is unable to bind GroES. E238 contributes to rapid GroES binding to GroEL because GroEL(E238A) is extremely sluggish in GroES binding. Polypeptide binding was not impaired by any mutations of E238A, T261A, and N265A. Rather, these mutants, especially GroEL(N265A), showed stronger polypeptide binding affinity than wild-type GroEL. Thus, these hydrophilic residues have a dual role; they help GroES binding on one hand but attenuate polypeptide binding on the other hand.

Chaperonin-affected refolding of alpha-lactalbumin: effects of nucleotides and the co-chaperonin GroES.

We have studied how nucleotides (ADP, AMP-PNP, and ATP) and the co-chaperonin GroES influence the GroEL-affected refolding of apo-alpha-lactalbumin. The refolding reactions induced by stopped-flow pH jumps were monitored by alpha-lactalbumin tryptophan fluorescence. The simple single-exponential character of the free-refolding kinetics of the protein allowed us to quantitatively analyze the kinetic traces of the GroEL-affected refolding with the aid of computer simulations, and to obtain the best-fit parameters for binding between GroEL and the refolding intermediate of alpha-lactalbumin by the non-linear least-squares method. When GroES was absent, the interaction between GroEL and alpha-lactalbumin could be well represented by a "cooperative-binding" model in which GroEL has two binding sites for alpha-lactalbumin with the affinity of the second site being tenfold weaker than that of the first, so that there is negative cooperativity between the two sites. The affinity between GroEL and alpha-lactalbumin was significantly reduced when ATP was present, while ADP and AMP-PNP did not alter the affinity. A comparison of this result with those reported previously for other target proteins suggests a remarkable adjustability of the GroEL 14-mer with respect to the nucleotide-induced reduction of affinity. When GroES was present, ATP as well as ADP and AMP-PNP were effective in reducing the affinity between GroEL and the refolding intermediate of alpha-lactalbumin. The affinity at a saturating concentration of ADP or AMP-PNP was about ten times lower than with GroEL alone. The ADP concentration at which the acceleration of the GroEL/ES-affected refolding of alphaLA was observed, was higher than the concentration at which the nucleotide-induced formation of the GroEL/ES complex took place. These results indicate that GroEL/ES complex formation itself is not enough to reduce the affinity for alpha-lactalbumin, and that further binding of the nucleotide to the GroEL/ES complex is required to reduce the affinity.

Refolding kinetics of staphylococcal nuclease and its mutants in the presence of the chaperonin GroEL.

We have analyzed the effect of the chaperonin GroEL on the refolding kinetics of staphylococcal nuclease and its three mutants by stopped-flow fluorescence measurements. It was found that a transient folding intermediate of staphylococcal nuclease was tightly bound to GroEL and refolded in the GroEL-bound state without releasing the non-native protein in solution, and the refolding rate in the GroEL-bound state was 0.01 s-1. The GroEL-affected refolding of the nuclease appears to be in decided contrast to that of apo-alpha-lactalbumin reported in our previous study, wherein alpha-lactalbumin was shown to be more weakly bound by GroEL and to refold in the free state in solution. In spite of the apparent difference between the proteins, the GroEL-affected refolding reactions of both the proteins can be represented by a common unified reaction scheme. On the basis of this scheme, the binding constant between the nuclease intermediate and GroEL was estimated to be larger than 10(9) M-1. The stoichiometry of binding of the nuclease and its mutants to GroEL was found to be two (nuclease/GroEL 14-mer). The increase in ionic strength resulted in a weakening of the interaction between the nuclease and GroEL, which was attributed to a weakening of the electrostatic attraction between the two proteins as a result of electrostatic screening by ions. Although ATP was found to accelerate the GroEL-affected refolding of the nuclease, the refolding rate was still far from the rate of the free refolding. The free refolding behavior of the nuclease and its mutants was restored in the presence of the cochaperonin GroES and ATP.