GroEL-mediated protein folding: making the impossible, possible

Abstract

Protein folding is a spontaneous process that is essential for life, yet the concentrated and complex interior of a cell is an inherently hostile environment for the efficient folding of many proteins. Some proteins-constrained by sequence, topology, size, and function-simply cannot fold by themselves and are instead prone to misfolding and aggregation. This problem is so deeply entrenched that a specialized family of proteins, known as molecular chaperones, evolved to assist in protein folding. Here we examine one essential class of molecular chaperones, the large, oligomeric, and energy utilizing chaperonins or Hsp60s. The bacterial chaperonin GroEL, along with its co-chaperonin GroES, is probably the best-studied example of this family of protein-folding machine. In this review, we examine some of the general properties of proteins that do not fold well in the absence of GroEL and then consider how folding of these proteins is enhanced by GroEL and GroES. Recent experimental and theoretical studies suggest that chaperonins like GroEL and GroES employ a combination of protein isolation, unfolding, and conformational restriction to drive protein folding under conditions where it is otherwise not possible.

Figure 1

The folding landscapes of large and topologically complex proteins are filled with kinetic traps. (A) Folding landscape of a small, rapidly folding protein with no requirement for folding assistance from a molecular chaperone. The vertical axis of the landscape captures the internal free energy of each protein conformation, while the width of the landscape is a measure of the configurational entropy of the protein at a particular energy level (Dill and Chan, 1997; Onuchic et al., 1997). (B) Folding landscape of a large and complex protein that depends upon molecular chaperones for productive folding. Spontaneous folding is inefficient or impossible, due to the prominent and deep local energy minima that easily trap the protein and lead to aggregation.

Figure 2

GroES binding to a GroEL ring creates an enclosed and enlarged cavity. (A) The molecular structure of unliganded GroEL (Braig et al., 1994; Braig et al., 1995) is illustrated as a space-filling model, with three subunits highlighted. The secondary structure of one subunit is shown, and for clarity this subunit is also illustrated alone, with the apical (‘a’), intermediate (‘i’) and equatorial (‘e’) domains highlighted. A cross-section of the unliganded GroEL barrel, illustrating the central cavity in each ring, is shown to the right. The substrate-binding apical surface is highlighted in the upper ring. Note that the apparent connection between the two cavities is an artifact of unresolved polypeptide density in the crystal structure and the two GroEL ring cavities are actually isolated from one another. (B) The molecular structure of the GroEL-ADP-GroES complex (Xu et al., 1997) as a space-filling model is illustrated. The secondary structure of one GroEL subunit from the cis ring is shown, along with a cross-section of the GroEL-GroES structure to the right. Note that the substrate-binding apical residues are again highlighted, but have been rotated away and are much less exposed to the cavity. Molecular structure images were created with PyMOL (DeLano, 2002).

Figure 3

The GroEL-GroES reaction cycle. The full GroEL reaction involves two half-cycles (a–d and e–h), where the assembly a GroEL-GroES cis complex on one ring is directly coupled to disassembly of a cis complex on the other ring. In (a), the open trans ring on a GroEL-ADP-GroES complex binds non-native substrate protein and ATP, triggering disassembly of the cis complex on the other ring (b). Rapid binding of GroES to the ATP and substrate occupied ring (c) encloses the substrate and releases the protein into the cis cavity. Subsequent hydrolysis of ATP within the cis complex to generate the GroEL-ADP-GroES complex primes the cis complex for disassembly and activates the trans ring for another round of ATP, substrate and GroES binding (d–f). The last two steps (g–h) then return the cycle to the starting point (shown dim). With each round of cis complex disassembly, the contents of the cavity are ejected into solution, including both folded and unfolded protein intermediates. A protein would typically remain within the enclosed cis cavity for 6–10 sec at 25°C.

Figure 4

GroEL can facilitate protein folding by either passive or active mechanisms. The dominant folding routes for an idealized, GroEL-dependent protein are shown. In the absence of GroEL, the protein can proceed to the native state via a productive, on-pathway intermediate state (I_on) or can form a misfolded and kinetically trapped off-pathway state (I_off) with no direct access to the native state. Both intermediates, as well as the unfolded ensemble (U), are highly prone to irreversible aggregation. GroEL could passively rescue a protein by binding I_on and U and blocking aggregation. Alternately, GroEL could actively enhance the folding of this protein by (1) binding and unfolding I_off, (2) binding U and I_on and preventing the formation of I_off, or (3) binding U and I_on and increasing the rate that N is produced.

Figure 5

Possible mechanisms of GroEL-induced protein unfolding. (A) Protein unfolding accomplished by thermodynamic coupling. A kinetically trapped folding intermediate that cannot bind to GroEL is in equilibrium with a less folded state that can bind to GroEL. In the presence of GroEL, mass action pulls the entire protein population into a GroEL-bound, unfolded state without affecting the intrinsic rate of unfolding. (B) Protein unfolding accomplished by catalytic unfolding. A kinetically trapped folding intermediate does not readily spontaneously unfold, but does bind to GroEL. The interaction of the folding intermediate with the hydrophobic GroEL ring results in a significant increase in the rate of unfolding. (C) Protein unfolding caused by forced unfolding. A kinetically trapped protein first binds to an open GroEL ring. Upon binding of ATP and GroES, the GroEL apical domains rearrange, resulting in significant stretching and unfolding of the bound substrate protein.

Figure 6

Stimulation of protein folding by repetitive unfolding and release: iterative annealing. A kinetically trapped protein is located in a deep energetic well that it cannot spontaneously escape (a). Upon binding to GroEL, the structure of the trapped intermediate is unfolded, inducing a displacement of the protein up the energy gradient of its folding landscape (b). Upon release from GroEL, the protein is afforded another chance to fold from a higher point on its energy landscape. Most of the protein falls back into the kinetic trap, though a fraction follows a productive route to the native state (c). GroEL rebinds the trapped protein and repeats the process until most of the population partitions to the native state.

Figure 7

Confinement of a non-native folding intermediate within the GroEL-GroES cavity results in smoothing of the protein’s energy landscape. The energy landscape of a kinetically trapped protein (a) is modified when the protein is enclosed in the GroEL-GroES cavity (b). Confinement of the non-native protein results in: (1) narrowing of the protein’s folding funnel by reducing the total number of allowed polypeptide conformation, (2) destabilization of expanded, kinetically trapped states that possess large radii of gyration incompatible with the volume of the GroEL-GroES cavity, (3) reduction in the height of transition state energy barriers that separate kinetically trapped states from productive folding pathways. The overall result of spatial confinement is a smoothing of the energy landscape of the non-native protein, opening productive folding routes that were not previously available (c).