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Chaperonin GroEL mediates the folding of protein encapsulated in a GroES-sealed cavity (cage). Recently, a critical role of negative charge clusters on the cage wall in folding acceleration was proposed based on experiments using GroEL single-ring (SR) mutants SR1 and SRKKK2 [Tang YC, et al. (2006) Cell 125:903-914; Chakraborty K, et al. (2010) Cell 142:112-122]. Here, we revisited these experiments and discovered several inconsistencies. (i) SR1 was assumed to bind to GroES stably and to mediate single-round folding in the cage. However, we show that SR1 repeats multiple turnovers of GroES release/binding coupled with ATP hydrolysis.(ii) Although the slow folding observed for a double-mutant of maltose binding protein (DMMBP) by SRKKK2 was attributed to mutations that neutralize negative charges on the cage wall, we found that the majority of DMMBP escape from SRKKK2 and undergo spontaneous folding in the bulk medium. (iii) An osmolyte, trimethylamine N-oxide, was reported to accelerate SRKKK2-mediated folding of DMMBP by mimicking the effect of cage-wall negative charges of WT GroEL and ordering the water structure to promote protein compaction. However, we demonstrate that in-cage folding by SRKKK2 is unaffected by trimethylamine N-oxide. (iv) Although it was reported that SRKKK2 lost the ability to assist the folding of ribulose-1,5-bisphosphate carboxylase/oxygenase, we found that SRKKK2 retains this ability. Our results argue against the role of the negative charges on the cage wall of GroEL in protein folding. Thus, in chaperonin studies, folding kinetics need to be determined from the fraction of the real in-cage folding.guanidium chloride | urea | fluorescence | anisotropy | apyrase T he bacterial GroEL/GroES chaperonin is an essential molecular chaperone that mediates the folding of various proteins (1, 2). GroEL consists of two rings stacked back to back, and each ring, made up of seven 57-kDa subunits, possesses a large central cavity. GroEL binds to a wide range of denatured proteins at the hydrophobic apical end of the central cavity to make a binary complex of GroEL/substrate protein. On binding of ATP to GroEL, GroES attaches to the apical end of the GroEL ring as a lid, generating a GroEL/GroES/substrate protein ternary complex, in which the substrate protein in the sealed cage starts folding. Hydrolysis of bound ATP triggers the detachment of the GroES lid to allow the substrate protein in the cage, whether folded or denatured, to be free. The next denatured protein is captured by GroEL, ATP binds to GroEL, and the cycle repeats. Single-round folding can be observed without the complication of coordinated ATP hydrolysis turnover of the two rings in GroEL by the use of a single-ring (SR) mutant of GroEL (SR1) that holds the GroES lid long enough for the substrate protein to finish folding in the cage (3).Two model mechanisms for the function of chaperonin in mediating protein folding have been proposed. The passive Anfinsen cage model explains that proteins fold in a spontaneous manner in the c...

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