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 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

Single-molecule spectroscopy of protein folding in a chaperonin cage

“…Even though our values for the rate constants (SI Appendix: Fig. S11) lie within the range of previous results obtained by enzymatic assays, a rigorous comparison to published results is complicated by the considerable spread of the rate constants reported (10,32,37,41,42). Possible reasons for this variability are the pronounced sensitivity of the system to solution conditions (10,37), temperature (Fig.…”

Requirement for binding multiple ATPs to convert a GroEL ring to the folding-active state

“…1B) because it allowed easy detection of this subunit in coexpression studies by its faster migration than I493C in SDS/ PAGE. 532⌬ exhibits a slightly reduced rate of steady-state ATP turnover relative to nondeleted GroEL, as expected based on recent observations that steady-state ATP turnover scales approximately with C terminus tail length of GroEL (23). If anything, EC3016 slightly stimulated the rate of steady-state turnover of both wildtype and 532⌬.…”

Multiple chaperonins in bacteria—novel functions and non-canonical behaviors

“…Recent studies on the C-terminus of GroEL have revealed a significant role of the C-terminal segment in the overall function of chaperonin (Tang et al 2006;Farr et al 2007), including interaction with the encapsulated substrate protein (Chen et al 2013). Moreover, genetic engineering experiments swapping GroEL tails from organisms growing in diverse temperatures indicated that the C-terminus of Fig.…”

“…Interestingly, expression of GroEL1 and GroEL3, but not GroEL2, is regulated by HrcA with the same proportion of identity to GGM (Table 1), indicating a possible correlation (Gould et al 2007a, b). Additionally, since alterations in GGM have been demonstrated to perturb GroEL's ATPase activity (Farr et al 2007) and that C-terminal functions as the thermometer (Nakamura et al 2004), lack of GGM-like tail in GroEL2 could explain the unusually high ATPase activity, thermal instability, and inability to functionally replace E. coli GroEL (George et al 2004). In addition, the observation that GroEL1 and GroEL3 possibly make hetero-oligomers adds strength to this hypothesis (Gould et al 2007a, b).…”