Directed Evolution of Substrate-Optimized GroEL/S Chaperonins

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...

Section: Discussionmentioning

confidence: 99%

Revisiting the contribution of negative charges on the chaperonin cage wall to the acceleration of protein folding

Motojima

Motojima-Miyazaki

Yoshida

2012

“…Alternatively, this observation could be explained by temperature-induced expression of chaperones, proteins that were found to mask phenotypic diversity in both prokaryotes and eukaryotes (39)(40)(41)(42)(43). The best-studied example is the eukaryotic heat shock protein 90 (Hsp90) that was shown to buffer phenotypic variation in several organisms.…”

Section: Discussionmentioning

confidence: 99%

Visualizing high error levels during gene expression in living bacterial cells

Meyerovich

Mamou

Ben-Yehuda

2010

To monitor inaccuracy in gene expression in living cells, we designed an experimental system in the bacterium Bacillus subtilis whereby spontaneous errors can be visualized and quantified at a single-cell level. Our strategy was to introduce mutations into a chromosomally encoded gfp allele, such that errors in protein production are reported in real time by the formation of fluorescent GFP molecules. The data reveal that the amount of errors can greatly exceed previous estimates, and that the error rate increases dramatically at lower temperatures and during stationary phase. Furthermore, we demonstrate that when facing an antibiotic threat, an increase in error level is sufficient to allow survival of bacteria carrying a mutated antibiotic-resistance gene. We propose that bacterial gene expression is error prone, frequently yielding protein molecules that differ slightly from the sequence specified by their DNA, thus generating a cellular reservoir of nonidentical protein molecules. This variation may be a key factor in increasing bacterial fitness, expanding the capability of an isogenic population to face environmental challenges.Bacillus subtilis | translational errors | variations in living cells | translation fidelity D NA is duplicated with remarkable fidelity to ensure that accurate genetic information is transmitted from one generation to the next. This information is passed from DNA to RNA and from RNA to protein during gene expression; however, the accuracy of these downstream events is relatively less understood. Although RNA and proteins are generally considered short-lived noninherited molecules, several diseases are now known to arise from errors occurring during transcription and translation (1-3), implying that faithful transfer of genetic information from DNA to proteins is crucial for maintaining proper cellular functions.

“…A precedent for improved folding by GroEL-GroES has been reported, with a directed-evolution experiment in which GroELGroES were genetically altered by a recombinational shuffling procedure carried out to optimize the extent of GFP folding in vivo (the rate could not be measured in this context) (29). Interestingly, the characterized mutant pair with the best refolding action (mutant 3-1) combined an equatorial domain substitution of GroEL that increased ATPase activity by 50% with a GroES mutant affecting a cavity-facing residue, Y71H.…”

Section: Influence Of Cavity Wall Vs Atpase On Folding By Groel-groesmentioning

confidence: 99%

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

Farr

Fenton

Horwich

2007

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 tailduplicated 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 Ϸ10% 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.chaperonin ͉ protein folding ͉ single ring