Kinetic Model for the Coupling between Allosteric Transitions in GroEL and Substrate Protein Folding and Aggregation

Abstract

The bacterial chaperonin GroEL and the co-chaperonin GroES assist in the folding of a number of structurally unrelated substrate proteins (SPs). In the absence of chaperonins, SP folds by the kinetic partitioning mechanism (KPM), according to which a fraction of unfolded molecules reaches the native state directly, while the remaining fraction gets trapped in a potentially aggregation-prone misfolded state. During the catalytic reaction cycle, GroEL undergoes a series of allosteric transitions (T<-->R-->R"-->T) triggered by SP capture, ATP binding and hydrolysis, and GroES binding. We developed a general kinetic model that takes into account the coupling between the rates of the allosteric transitions and the folding and aggregation of the SP. Our model, in which the GroEL allosteric rates and SP-dependent folding and aggregation rates are independently varied without prior assumption, quantitatively fits the GroEL concentration-dependent data on the yield of native ribulose bisphosphate carboxylase/oxygenase (Rubisco) as a function of time. The extracted kinetic parameters for the GroEL reaction cycle are consistent with the available values from independent experiments. In addition, we also obtained physically reasonable parameters for the kinetic steps in the reaction cycle that are difficult to measure. If experimental values for GroEL allosteric rates are used, the time-dependent changes in native-state yield at eight GroEL concentrations can be quantitatively fit using only three SP-dependent parameters. The model predicts that the differences in the efficiencies (as measured by yields of the native state) of GroEL, single-ring mutant (SR1), and variants of SR1, in the rescue of mitochondrial malate dehydrogenase, citrate synthase, and Rubisco, are related to the large variations in the allosteric transition rates. We also show that GroEL/S mutants that efficiently fold one SP at the expense of all others are due to a decrease in the rate of a key step in the reaction cycle, which implies that wild-type GroEL has evolved as a compromise between generality and specificity. We predict that, under maximum loading conditions and saturating ATP concentration, the efficiency of GroEL (using parameters for Rubisco) depends predominantly on the rate of R-->R" transition, while the equilibrium constant of the T<-->R has a small effect only. Both under sub- and superstoichiometric GroEL concentrations, enhanced efficiency is achieved by rapid turnover of the reaction cycle, which is in accord with the predictions of the iterative annealing mechanism. The effects are most dramatic at substoichiometric conditions (most relevant for in vivo situations) when SP aggregation can outcompete capture of SP by chaperonins.

Reconciling theories of chaperonin accelerated folding with experimental evidence

“…Active models IAM via ATP The traditional iterative annealing model (IAM) says that the ATP-driven cycles of binding and unbinding to GroEL accelerate folding by periodically disrupting or destabilizing off-pathway misfolded states [9,10,22,72,73,77,80,[102][103][104][105][106][107][108][109][110][111][112][113][114]. We suggest that GroEL may be able to accomplish this without releasing substrates into the cytosol (the stationary IAM).…”

“…Simple kinetics models of GroEL/ES behavior assume that the entire time a protein is bound to GroEL it is either able to continue folding [148], or immobilized [113]. In reality, proteins may spend a fraction of their time with GroEL mobile or immobilized.…”

Minimal Models for Proteins and RNA: From Folding to Function

“…8,9,122 In the cellular environment molecular chaperones, such as trigger factor 123 or the GroEL-GroES chaperonin system powered by ATP molecules, 93 increase the yield of the native state for substrate proteins that are prone to misfold. 93,124 Thus, the normal operation of chaperonin systems are crucial to cellular function. The most well studied chaperonin is GroEL, that has two heptameric rings, stacked back-to-back.…”

Generalized iterative annealing model for the action of RNA chaperones

“…Physically, this situation is not that dissimilar to the role mini chaperone (apical domain of GroEL) plays in annealing certain non-stringent substrates [37]. In the absence of RNA chaperone, the KPM predicts that the initial pool of unfolded RNA ribozymes are partitioned into folded and misfolded conformations, described by the following set of rate equations [43].…”

“…Physically, this situation is not that dissimilar to the role mini chaperone (apical domain of GroEL) plays in annealing certain non-stringent substrates [37]. In the absence of RNA chaperone, the KPM predicts that the initial pool of unfolded RNA ribozymes are partitioned into folded and misfolded conformations, described by the following set of rate equations [43].For a given ribozyme concentration, a fraction φ F of ribozyme folds into the native state directly at a rate k F , and the remaining fraction (1 − φ F ) is misfolded upon undergoing non-specific collapse transition. The solution of Eq.4, in terms of the probability of not being folded, is given by P U +M (t) ≈ φ F e −k F t + (1 − φ F )e −k S t , which follows from the KPM…”