Determination of the Number of Active GroES Subunits in the Fused Heptamer GroES Required for Interactions with GroEL

Abstract

A double-heptamer ring chaperonin GroEL binds denatured substrate protein, ATP, and GroES to the same heptamer ring and encapsulates substrate into the central cavity underneath GroES where productive folding occurs. GroES is a disk-shaped heptamer, and each subunit has a GroEL-binding loop. The residues of the GroEL subunit responsible for GroES binding largely overlap those involved in substrate binding, and the mechanism by which GroES can replace the substrate when GroES binds to GroEL/substrate complex remains to be clarified. To address this question, we generated single polypeptide GroES by fusing seven subunits with various combinations of active and GroEL binding-defective subunits. Functional tests of the fused GroES variants indicated that four active GroES subunits were required for efficient formation of the stable GroEL/ GroES complex and five subunits were required for the productive GroEL/substrate/GroES complex. An increase in the number of defective GroES subunits resulted in a slowing of encapsulation and folding. These results indicate the presence of an intermediate GroEL/substrate/GroES complex in which the substrate and GroES bind to GroEL by sharing seven common binding sites.

Effects of C-terminal Truncation of Chaperonin GroEL on the Yield of In-cage Folding of the Green Fluorescent Protein

“…To demonstrate this hypothesis, the N265A mutant of GroEL (a substrate trap mutant) was added to the reaction mixture. The substrate trap mutant has been shown to bind a denatured protein more strongly than wild-type GroEL even in the presence of ATP, although it does not bind GroES (27,28). When an excess amount of the substrate trap mutant was present in the refolding mixture of SR1⌬C/ES/GFP, the yield of GFP folding was markedly decreased (Fig.…”

Probing Open Conformation of GroEL Rings by Cross-linking Reveals Single and Double Open Ring Structures of GroEL in ADP and ATP

“…Mutant proteins were generated by site-directed mutagenesis using the Prime-STAR mutagenesis basal kit from Takara. GroEL mutants and GroES were purified as described previously (25,26). GroEL with mutations A384C/S509C/C138A/C458A/C519A (27) (termed GroEL(2Cys)) was stored in HKM buffer (25 mM HEPES-KOH, pH 7.4, 5 mM MgCl 2 , and 100 mM KCl) containing 5 mM dithiothreitol.…”

Protein folding and aggregation in bacteria

“…The region of substrate and GroES binding at the apical domain of GroEL largely overlaps, but the mechanism by which GroES competes with the substrate in the GroEL-substrate complex is still unclear. Recent studies suggest the existence of an intermediate GroEL-substrate-GroES complex in which the substrate and GroES bind to GroEL by sharing seven common binding sites [83].…”