Coupling between protein folding and allostery in the GroE chaperonin system

Abstract

GroEL is an allosteric protein that facilitates protein folding in an ATP-dependent manner. Herein, the relationship between cooperative ATP binding by GroEL and the kinetics of GroE-assisted folding of two substrates with different GroES dependence, mouse dihydrofolate reductase (mDHFR) and mitochondrial malate dehydrogenase, is examined by using cooperativity mutants of GroEL. Strong intra-ring positive cooperativity in ATP binding by GroEL decreases the rate of GroEL-assisted mDHFR folding owing to a slow rate of the ATP-induced transition from the protein-acceptor state to the protein-release state. Inter-ring negative cooperativity in ATP binding by GroEL is found to affect the kinetic partitioning of mDHFR, but not of mitochondrial malate dehydrogenase, between folding in solution and folding in the cavity underneath GroES. Our results show that protein folding by this "two-stroke motor" is coupled to cooperative ATP binding.

Figure 1

Effect of intra-ring positive cooperativity in GroEL on the rate of GroEL-mediated folding of mDHFR. Values of the rates of refolding (k_f) of mDHFR in the presence of different GroEL mutants were determined as described in Experimental Procedures. They are plotted against the values of the Hill coefficients (n_H) (A) and rates (k₁) of the TT → TR conformational transitions (B) of the respective GroEL mutants, which were determined previously (23). The values of the correlation coefficient (r) are 0.98 (A) and 0.96 (B). The errors are 1% in k_f and about 5% in k₁.

Figure 2

Rates of GroEL-mediated folding of mDHFR as a function of the extent of negative cooperativity. Values of the rates of refolding (k_f) of mDHFR, in the presence of different GroEL mutants and in the absence (○) or presence of 250 nM (●) or 500 nM (■) GroES oligomer, were determined as described in Experimental Procedures. Values of the allosteric equilibrium constants L₁ (which equals [TR]/[TT]) and L₂ (which equals [RR]/[TR]) were determined by fitting data of initial rates of ATPase activity as a function of ATP concentration as described (11). Values of logk_f are plotted against the values of log(L₁/L₂), which are measures of the extent of negative cooperativity (11). The value of the correlation coefficient (r) is 0.99 for each of the three linear fits. The errors are up to 1% in k_f and about 10% in log(L₁/L₂).

Figure 3

Rates of GroEL-mediated folding of mitochondrial MDH as a function of the extent of negative cooperativity. Values of the rates of refolding (k_f) of MDH, in the presence of different GroEL mutants and in the absence (○) or presence of 250 nM (●) or 500 nM (■) GroES oligomer, were determined as described in Experimental Procedures. Values of logk_f are plotted against the values of log(L₁/L₂), which were calculated by fitting data of initial rates of ATPase activity as a function of ATP concentration as described (11). The errors are up to 10% in k_f and about 10% in log(L₁/L₂).