How protein thermodynamics and folding mechanisms are altered by the chaperonin cage: molecular simulations

Abstract

How the Escherichia coli GroEL/ES chaperonin assists folding of a substrate protein remains to be uncovered. Recently, it was suggested that confinement into the chaperonin cage itself can significantly accelerate folding of a substrate. Performing comprehensive molecular simulations of eight proteins confined into various sizes L of chaperonin-like cage, we explore how and to what extent protein thermodynamics and folding mechanisms are altered by the cage. We show that a substrate protein is remarkably stabilized by confinement; the estimated increase in denaturation temperature DeltaTf is as large as approximately 60 degrees C. For a protein of size R0, the stabilization DeltaTf scales as (R0/L)nu, where nu approximately 3, which is consistent with a mean field theory of polymer. We also found significant free energy cost of confining a protein, which increases with R0/L, indicating that the confinement requires external work provided by the chaperonin system. In kinetic study, we show the folding is accelerated in a modestly well confined case, which is consistent with a recent experimental result on ribulose-1,5-bisphosphate carboxylase-oxygenase folding and simulation results of a beta hairpin. Interestingly, the acceleration of folding is likely to be larger for a protein with more complex topology, as quantified by the contact order. We also show how ensemble of folding pathways are altered by the chaperonin-like cage calculating a variant of value used in the study of spontaneous folding.

Fig. 1.

Cartoon of chaperonin-like cage and proteins studied. (Left) The chaperonin-like cage is modeled as a cylindrical box with a characteristic length L, in which a folding protein molecule is confined. (Right) The native structures of five proteins studied (of eight); they are, from the top left to the bottom right, protein G, 434 repressor, src SH3 domain, Ada2h, and fibronectin type 3. The Protein Data Bank codes are given above the structures, and the number of residues and radii of gyration in the native structures are shown in parentheses.

Fig. 2.

Folding time course and distribution of the nativeness measure Q both for unconfined (Left) and confined (Right) protein G (Protein Data Bank code 2gb1) at the transition temperature of unconfined system T⁰_f.(Top) Time series Q(t) are plotted for representative trajectories. (Middle) Scattered plots in the (Q, R_g) plane are drawn from the same trajectories. (Bottom) The free energy profile F(Q) as a function of Q is plotted. The confinement induces restriction to the denatured distribution as seen (Middle Right), which makes the denatured state less stable (Bottom Right) (at T = T⁰_f).

Fig. 3.

Change in the thermodynamic stability of a chaperonin-caged protein. (a) The heat capacity C_v of protein G (Protein Data Bank ID code 2gb1) as a function of temperature T for several different cage sizes L [L = ∞ (bulk), 50, 30, 20, and 15 Å]. As the cage size decreases, the folding transition temperature T_f is increased and the peak becomes broader. (b) The relative change in the folding transition temperatures (T_f – T⁰_f)/T⁰_f caused by confinement plotted against N^3/5/L for five proteins studied. Both axes are in the logarithmic scale. Symbols are defined in the figure. The straight line is obtained by the linear regression.

Fig. 4.

Estimated free energy cost of confining a protein molecule into the chaperonin-like cage plotted against N^3/5/L. Results for five proteins are depicted, and symbols used are the same as those in Fig. 3.

Fig. 5.

(a) Folding rate constants k_f of eight proteins unconfined and confined in the chaperonin cage against the relative contact order (RCO) × N^0.607 (slope: –2.19 ± 0.43 for bulk, –1.39 ± 0.45 for L = 25 Å). (b) Folding rate constant k_f as a function of N^3/5/L for five proteins. Each k_f is normalized by the folding rate constant in the bulk system .

Fig. 6.

Schematic view of protein folding funnel of bulk and confined proteins. The conformational entropy of the denatured state decreases with the confinement factor N^3/5/L, which leads to a steeper funnel. For the confinement factor larger than N^3/5/L ≈0.7, the energy landscape becomes increasingly more rugged, and thus folding becomes slower.