Chaperonin chamber accelerates protein folding through passive action of preventing aggregation

Abstract

The original experiments reconstituting GroEL-GroES-mediated protein folding were carried out under "nonpermissive" conditions, where the chaperonin system was absolutely required and substrate proteins could not achieve the native state if diluted directly from denaturant into solution. Under "permissive" conditions, however, employing lower substrate concentration and lower temperature, some substrate proteins can be refolded both by the chaperonin system and while free in solution. For several of these, the protein refolds more rapidly inside the GroEL-GroES cis chamber than free in solution, suggesting that the chamber may have an active role in assisting protein folding. Here, we observe that the difference is caused by reversible multimolecular association while folding in solution, an avenue of kinetic partitioning that slows the overall rate of renaturation relative to the chaperonin chamber, where such associations cannot occur. For Rubisco, reversible aggregation during folding in solution was observed by gel filtration. For a mutant of maltose-binding protein (DM-MBP), the rate of folding in solution declined with increasing concentration, and the folding reaction produced light scattering. Under solution conditions where chloride was absent, however, light scattering no longer occurred, and DM-MBP folded at the same rate as in the cis cavity. In a further test, dihydrofolate reductase, thermally inactivated in the cis cavity or in solution, was substantially reactivated upon temperature downshift in the cis cavity but not in solution, where aggregation occurred. We conclude that the GroEL-GroES chamber behaves as a passive "Anfinsen cage" whose primary role is to prevent multimolecular association during folding.

Fig. 1.

Renaturation of Rubisco under permissive conditions while free in solution is slower than chaperonin-mediated refolding and is associated with aggregation. (A) Rubisco refolding under permissive conditions (80 nM final concentration and 15 °C), monitored by recovery of enzymatic activity either free in solution (spont), with or without 7.5 μM BSA, or as mediated by the chaperonin systems, SR1–GroES [a stable cis folding chamber (9)] or cycling GroEL–GroES, with or without 7.5 μM BSA. Apparent rate constants are indicated for the respective refolding reactions. Note that BSA does not affect the rate of the chaperonin-mediated renaturation, whereas it has a major effect on refolding free in solution. Even so, the reaction in solution containing BSA is 5- to 10-fold slower than the chaperonin-mediated reactions. Note that because native Rubisco is a homodimer, the encapsulated monomers in the SR1–GroES reaction had to be released from the complex to allow folded ones to assemble, accomplished by a brief 4 °C incubation that releases GroES (see ref. 11) before enzyme assay. (B) Dynamic light-scattering measurements during refolding of 80 nM Rubisco free in solution in the presence of 7.5 μM BSA, reported in arbitrary units (red). As a control, light scattering by the same amount of native Rubisco recovered in the reaction was measured in the presence of the same concentration of BSA, producing negligible scattering (blue). BSA alone in buffer also produced negligible scattering (black). (C) Gel filtration analyses of 80 nM [³⁵S]Rubisco carried out at 3 times during its refolding free in solution in the presence of 7.5 μM BSA. Amounts of radioactive Rubisco recovered in fractions from a Superose 6 column are reported as percentages of the input Rubisco. The void volume peak at 6 mL corresponds to ≈10 MDa. The peak at 17 mL corresponds to native Rubisco homodimer, 102 kDa, established by independent gel filtration analysis of native Rubisco (dotted line). Note that species in the 10-min experiment that are intermediate in size between the void peak and native appear to be converted at later times into the native species and into additional void volume species, apparently reflecting, respectively, reversal of aggregation to produce the native state and irreversible aggregation.

Fig. 2.

Renaturation of DM-MBP under permissive conditions is slower free in solution than chaperonin-mediated, associated with aggregation occurring free in solution as indicated by concentration dependence of rate of refolding and dynamic light scattering. (A) DM-MBP free in solution (black; spont) at 100 nM refolds at a rate that is only ≈1/10 the rate of the chaperonin reaction mediated by GroEL–GroES–ATP or by SR1–GroES–ATP (red and blue). Renaturation is monitored by an increase in tryptophan fluorescence intensity (excitation, 295 nm; emission, 345 nm), as described in Methods. (B) Rate of refolding free in solution is inversely related to the concentration of DM-MBP. (C) Dynamic light scattering, in arbitrary units, during refolding of 100 nM DM-MBP in solution. In contrast with the double mutant, wild-type MBP did not produce significant light scattering when refolding free in solution. (D) Wild-type MBP, 100 nM, does not exhibit concentration dependence of its refolding rate.

Fig. 3.

Relief of light scattering behavior of 100 nM DM-MBP, folding free in solution, by deletion of chloride from the refolding buffer is associated with restoration of folding rate to that of the chaperonin-mediated reaction. (A) Light scattering showing that deletion of chloride from the folding buffer (replacing it with acetate) relieves light-scattering behavior of DM-MBP folding free in solution. (B) Refolding of DM-MBP free in chloride-deficient solution measured by the increase of tryptophan fluorescence intensity, showing that the rate of renaturation in chloride-deficient solution (black) is, under these conditions, the same as that of the chaperonin reactions mediated by GroEL–GroES–ATP (red) or SR1–GroES–ATP (blue), also in chloride-free conditions.

Fig. 4.

Comparison of thermal inactivation of an already-native monomeric protein, DHFR, free in solution and inside a stable cis cavity, and recovery of activity after temperature downshift in the cis cavity but not in solution. (A and B) Thermal inactivation. Native DHFR free in solution or in a cis ternary GroEL–GroES–ADP-aluminum fluoride complex was exposed to elevated temperatures for 10 min, and enzymatic assay was then immediately carried out at 23 °C. The loss of enzymatic activity with increasing temperature occurred to a nearly identical extent while free in solution (Solution) or while in the cis cavity (cis). (See Methods for formation of the cis ternary complex and SI Methods for a test that assured that the cis complex remained stable during thermal treatment.) (C and D) Recovery from thermal inactivation. After inactivation of DHFR at 50 °C (middle bars), the mixture was returned to 23 °C for 30 min and then assayed for enzymatic activity. A substantial amount of activity was recovered from the cis complex (D, right bar) but not from the solution reaction (C, right bar), where aggregation had occurred (see Results and Fig. S2).