Perturbed ATPase activity and not "close confinement" of substrate in the cis cavity affects rates of folding by tail-multiplied GroEL

Abstract

Folding of substrate proteins inside the sequestered and hydrophilic GroEL-GroES cis cavity favors production of the native state. Recent studies of GroEL molecules containing volume-occupying multiplications of the flexible C-terminal tail segments have been interpreted to indicate that close confinement of substrate proteins in the cavity optimizes the rate of folding: the rate of folding of a larger protein, Rubisco (51 kDa), was compromised by multiplication, whereas that of a smaller protein, rhodanese (33 kDa), was increased by tail duplication. Here, we report that this latter effect does not extend to the subunit of malate dehydrogenase (MDH), also 33 kDa. In addition, single-ring versions of tail-duplicated and triplicated molecules, comprising stable cis complexes, did not produce any acceleration of folding of rhodanese or MDH, nor did they show significant retardation of the folding of Rubisco. Tail quadruplication produced major reduction in recovery of native protein with both systems, the result of strongly reduced binding of all three substrates. When steady-state ATPase of the tail-multiplied double-ring GroELs was examined, it scaled directly with the number of tail segments, with more than double the normal ATPase rate upon tail triplication. As previously observed, disturbance of ATPase activity of the cycling double-ring system, and thus of "dwell time" for the folding protein in the cis cavity, produces effects on folding rates. We conclude that, within the limits of the approximately 10% decrease of cavity volume produced by tail triplication, there does not appear to be an effect of "close confinement" on folding in the cis cavity.

Fig. 1.

No acceleration of rhodanese refolding by tail-multiplied variants of SR1. Guanidine-HCl-denatured rhodanese was diluted into buffer containing the indicated chaperonin to form a binary complex. GroES and ATP were added, and at the indicated times, the recovery of native protein was measured by assay of rhodanese enzymatic activity. The amount of native rhodanese recovered is expressed as a percentage of the total input rhodanese.

Fig. 2.

No acceleration of MDH refolding by tail-multiplied versions of either SR1 or GroEL. MDH denatured in guanidine-HCl was diluted into buffer containing the indicated single-ring (A) or double-ring (B) chaperonin to form binary complex. GroES and ATP were added, and at the indicated times, the recovery of native MDH was measured by assay of MDH enzyme activity. The yield of MDH activity recovered is expressed as a percentage of the initial MDH added to the reaction.

Fig. 3.

Rate of Rubisco refolding is not affected by tail-duplication or triplication in SR1. Guanidine-HCl-denatured Rubisco was diluted into buffer containing the indicated chaperonin. GroES and ATP were added, and the amount of native Rubisco recovered at the indicated times was determined by enzymatic assay. The recoveries are expressed as a percentage of the total input Rubisco.

Fig. 4.

Tail quadruplication greatly reduces substrate binding to both SR1 and GroEL. (A) Rubisco binding. (B) MDH binding. (C) Rhodanese binding. (D) Rhodanese binding and encapsulation. ³⁵S-labeled Rubisco (A), MDH (B), or rhodanese (C and D) were denatured in guanidine-HCl and diluted into buffer containing the indicated chaperonin. In A–C, binary complexes were purified by gel-filtration chromatography, and the amount of substrate protein recovered was compared with that for GroEL (filled bars) or SR1 (open bars). In D, GroES and ATP were added before gel filtration to encapsulate bound ³⁵S-rhodanese, and gel-filtration was carried out in the presence of 1 mM ADP. Error bars in C represent the standard deviation for three experiments.

Fig. 5.

The steady-state ATPase activity of GroEL and GroEL-GroES scales with the number of C-terminal tails, whereas GroES completely suppresses the activity of tail-multiplied SR1. The rate of ATP hydrolysis for tail-multiplied versions of GroEL (A) and SR1 (B) was determined by the malachite green assay in the absence (filled bars) or presence (open bars) of GroES, using 1 mM ATP. Error bars represent the standard deviation in three experiments.