Iterative annealing mechanism explains the functions of the GroEL and RNA chaperones

Abstract

Molecular chaperones are ATP-consuming biological machines, which facilitate the folding of proteins and RNA molecules that are kinetically trapped in misfolded states for long times. Unassisted folding occurs by the kinetic partitioning mechanism according to which folding to the native state, with low probability as well as misfolding to one of the many metastable states, with high probability, occur rapidly on similar time scales. GroEL is an all-purpose stochastic machine that assists misfolded substrate proteins (SPs) to fold. The RNA chaperones help the folding of ribozymes that readily misfold. GroEL does not interact with the folded proteins but CYT-19 disrupts both the folded and misfolded ribozymes. Despite this major difference, the Iterative Annealing Mechanism (IAM) quantitatively explains all the available experimental data for assisted folding of proteins and ribozymes.Driven by ATP binding and hydrolysis and GroES binding, GroEL undergoes a catalytic cycle during which it samples three allosteric states, referred to as T (apo), R (ATP bound), and R (ADP bound).In accord with the IAM predictions, analyses of the experimental data shows that the efficiency of the GroEL-GroES machinery and mutants is determined by the resetting rate k R →T , which is largest for the wild type GroEL. Generalized IAM accurately predicts the folding kinetics of Tetrahymena ribozyme and its variants. Chaperones maximize the product of the folding rate and the steady state native state fold by driving the substrates out of equilibrium. Neither the absolute yield nor the folding rate is optimized.tion of the SPs with GroEL in the T state. We present a schematic in Fig. 3b of the reaction cycle in a single ring. We ought to emphasize, right at the outset, that recent advances show that when challenged with SPs the functioning state is the symmetric 14-mer GroEL-GroEs complex, resembling a football 18,19 (see Fig.2b), and not the asymmetric bullet structure as had been thought for a long time.The parts list of this complex machine is GroEL, GroES, MgATP, and the SPs, that require assistance to reach the folded structures. A few words about the SPs are in order. It has been shown long ago that GroEL is a promiscuous machine that interacts with a vast majority of E. Coli. proteins as long as they are presented in the misfolded states 20 . This observation and the subsequent demonstration that most of the SPs used to study GroEL assisted folding are ones not found in E. Coli. further buttresses this point. For discussion purposes, we distinguish between permissive and non-permissive conditions. Under permissive conditions, folding to the native state occurs readily in vitro on a biologically relevant time scale (τ B ), 4which is between 20-40 minutes for E. coli proteins at 37 • C. Under non-permissive conditions, spontaneous folding does not occur in vitro with sufficient yield of the folded protein on the time scale, τ B . The SPs satisfying this criterion are deemed to be stringent substrates for GroEL. Several in vitro exp...

Measuring protein stability in the GroEL chaperonin cage reveals massive destabilization

“…Finally, it is important to note that our finding that the cavity environment is destabilizing supports the iterative annealing mechanism of action proposed for the chaperonin GroEL ( Todd et al, 1996 ; Thirumalai et al, 2020 ). According to this mechanism, protein substrates undergo kinetic partitioning, during each reaction cycle of GroEL, between productive folding to the native state and mis-folding.…”

Recent advances in understanding catalysis of protein folding by molecular chaperones

“…Chaperonins (also referred to as Hsp60s) are large oligomeric complexes that function as nanocages for single protein molecules to fold in isolation [47–51]. They participate in folding ~ 10% of the cytosolic proteome, including essential proteins that fail to reach their native state spontaneously and cannot utilize other chaperone systems [52–56].…”

Retardation of folding rates of substrate proteins in the nanocage of GroEL

“…Of relevance here is the implication that folding rates per se could be accelerated (modestly) or even retarded relative to the bulk. The IAM quantitatively explains the results of a large number of experiments 12 , including observations that mutations in GroEL render it less efficient than the wild type 12 . Recent experiments [32][33] have also established that in the presence of SP the chaperonin machinery responds rapidly by processing folding in both the chambers (GroEL/ES is a parallel processing machine), a discovery that accords well the IAM predictions.…”

“…In the full chaperonin machinery, the residence time of the SP in the expanded internal cage of GroEL is just a few seconds, and not 300 minutes 12 . Nevertheless, we find using the cycling system consisting of GroES, GroEL, and ATP that the folding rate of rhodanese is retarded relative to its value in the bulk.…”

“…The cavity volume increases roughly from 85,000Å 3 in the T state to about 170,000Å 3 in the R I or R II state 11 . In the R II state nucleotide state, the cavity can fully encapsulate a protein with a maximum of ∼ 500 amino acid residues 12 .…”