Molecular Chaperones Assist Protein Folding
Proteins begin to fold as they are being synthesized, so the renaturation of a denatured protein in vitro may not entirely mimic the folding of a protein in vivo. In addition, proteins fold in vivo in the presence of extremely high concentrations of other proteins with which they can potentially interact. Molecular chaperones are essential proteins that bind to unfolded and partially folded polypeptide chains to disrupt the improper association of exposed hydrophobic segments that would otherwise lead to non-native folding as well as polypeptide aggregation and precipitation.
In essence, molecular chaperones function to lift folding polypeptides out of the false minima in their folding funnels. This is especially important for multidomain and multisubunit proteins, whose components must fold fully before they can properly associate with each other.
Many molecular chaperones were first described as heat shock proteins (Hsp) because their rate of synthesis is increased at elevated temperatures. Presumably, the additional chaperones are required to recover heat-denatured proteins or to prevent misfolding under conditions of environmental stress.
Cells Contain a Variety of Molecular Chaperones. There are several classes of molecular chaperones in both prokaryotes and eukaryotes, including the following:
1. The Hsp70, a family of highly conserved 70-kD proteins in both prokaryotes and eukaryotes. In association with the cochaperone protein Hsp40, they facilitate the folding of newly synthesized proteins and reverse the denaturation and aggregation of proteins. Hsp70 proteins also function to unfold proteins in preparation for their transport through membranes and to subsequently refold them.
2. Trigger factor, a ribosome-associated chaperone in prokaryotes that prevents the aggregation of polypeptides as they emerge from the ribosome. Trigger factor and Hsp70 are the first chaperones a newly made prokaryotic protein encounters. Subsequently, many partially folded proteins are handed off to other chaperones to complete the folding process. E. coli can tolerate the elimination of trigger factor or Hsp70, but not both, thereby indicating that they are functionally redundant. Eukaryotes lack trigger factor but contain other small chaperones that have similar functions.
3. The chaperonins, which form large, multi subunit, cage like assemblies in both prokaryotes and eukaryotes. They bind improperly folded proteins and induce them to refold inside an internal cavity.
4. The Hsp90, a family of 90-kD eukaryotic proteins that mainly facilitate the late stages of folding of proteins involved in cellular signaling. Hsp90 proteins are among the most abundant proteins in eukaryotes, accounting for up to 6% of cellular protein under stressful conditions that destabilize proteins.
Most Chaperones Require ATP. All molecular chaperones operate by binding to an unfolded or aggregated protein’s solvent-exposed hydrophobic surface and subsequently releasing it. The disruption of intramolecular and/or intermolecular aggregates by chaperone binding permits the substrate protein to continue folding after its release. In most cases, this bind-and-release process is repeated several times before the substrate protein reaches its native state, whereupon it no longer has the exposed hydrophobic surfaces to which chaperones bind. Most molecular chaperones are ATPases; that is, enzymes that catalyze the hydrolysis of ATP (adenosine triphosphate) to ADP (adenosine diphosphate) and Pi (inorganic phosphate):
ATP + H2O → ADP + Pi
The ATP complex of the chaperone binds the unfolded or aggregated substrate protein, whereas its ADP complex releases it. Thus, the favorable free energy change of ATP hydrolysis drives the chaperone’s bind-and-release reaction cycle.
The GroEL/ES Chaperonin Forms Closed Chambers in Which Proteins Fold
The chaperonins in E. coli consist of two types of subunits named GroEL and GroES. The X-ray structure of a GroEL–GroES–(ADP)7 complex, determined by Arthur Horwich and Paul Sigler, reveals 14 identical 549-residue GroEL subunits arranged in two stacked rings of seven subunits each. This complex is capped at one end by a domelike heptameric ring of 97-residue GroES subunits to form a bullet-shaped complex with C7 symmetry. The two GroEL rings each enclose a central chamber with a diameter of ∼45 Å in which partially folded proteins fold to their native conformations. A barrier in the center of the complex prevents a folding protein from passing between the two GroEL chambers. The GroEL ring that contacts the GroES heptamer is called the cis ring; the opposing GroEL ring is known as the trans ring.
ATP Binding and Hydrolysis Drive the Conformational Changes in GroEL/ES
Each GroEL subunit has a binding pocket for ATP that catalyzes the hydrolysis of its bound ATP to ADP + Pi. When the cis ring subunits hydrolyze their bound ATP molecules and release the product Pi, the protein undergoes a conformational change that widens and elongates the cis inner cavity so as to more than double its volume from 85,000 Å3 to 175,000 Å3. The expanded cavity can enclose a partially folded substrate protein of at least 70 kD. All seven subunits of the GroEL ring act in concert; that is, they are mechanically linked such that they change their conformations simultaneously.
The cis and trans GroEL rings undergo conformational changes in a reciprocating fashion, with events in one ring influencing events in the other ring. The entire GroEL/ES chaperonin complex functions as follows.
1. One GroEL ring that has bound 7 ATP also binds an improperly folded substrate protein, which associates with hydrophobic patches that line the inner wall of the GroEL chamber. The GroES cap then binds to the GroEL ring like a lid on a pot, inducing a conformational change in the resulting cis ring that buries the hydrophobic patches, thereby depriving the substrate protein of its binding sites. This releases the substrate protein into the now enlarged and closed cavity, where it commences folding. The cavity, which is now lined only with hydrophilic groups, provides the substrate protein with an isolated microenvironment that prevents it from nonspecifically aggregating with other misfolded proteins. Moreover, the conformational change that buries GroEL’s hydrophobic patches stretches and thereby partially unfolds the improperly folded substrate protein before it is released. This rescues the substrate protein from a local energy minimum in which it had become trapped thereby permitting it to continue its conformational journey down the folding funnel toward its native state (the state of lowest free energy).
2. Within, ∼10 s (the time the substrate protein has to fold), the cis ring catalyzes the hydrolysis of its 7 bound ATPs to ADP + Pi and the Pi is released. The absence of ATP’s γ phosphate group weakens the interactions that bind GroES to GroEL.
3. A second molecule of improperly folded substrate protein binds to the trans ring followed by 7 ATP. Conformational linkages between the cis and trans rings prevent the binding of both substrate protein and ATP to the trans ring until the ATP in the cis ring has been hydrolyzed.
4. The binding of substrate protein and ATP to the trans ring conformationally induces the cis ring to release its bound GroES, 7 ADP, and the presumably now better-folded substrate protein. This leaves ATP and substrate protein bound only to the trans ring of GroEL, which now becomes the cis ring as it binds GroES.
Steps 1 through 4 are then repeated. The GroEL/ES system expends 7 ATPs per folding cycle. If the released substrate protein has not achieved its native state, it may subsequently rebind to GroEL (a substrate protein that has achieved its native fold lacks exposed hydrophobic groups and hence cannot rebind to GroEL). Typically, only ∼5% of substrate proteins fold to their native state in each reaction cycle. Thus, to fold half the substrate protein present would require log(1 − 0.5)/log(1 − 0.05) ≈ 14 reaction cycles and hence 7 × 14 = 98 ATPs (which appears to be a profligate use of ATP but constitutes only a small fraction of the thousands of ATPs that must be hydrolyzed to synthesize a typical polypeptide and its component amino acids). Because protein folding occurs alternately in the two GroEL rings, the proper functioning of the chaperonin requires both GroEL rings, even though their two cavities are unconnected.
Experiments indicate that the GroEL/ES system interacts with only a subset of E. coli proteins, most with molecular masses in the range 20 to 60 kD. These proteins tend to contain two or more α/β domains that mainly consist of open β sheets. Such proteins are expected to fold only slowly to their native state because the formation of hydrophobic sheets requires a large number of specific longrange interactions. Proteins dissociate from GroEL/ES after folding, but some frequently revisit the chaperonin, apparently because they are structurally labile or prone to aggregate and must return to GroEL for periodic maintenance. Eukaryotic cells contain the chaperonin TRiC, with double rings of eight nonidentical subunits, each of which resembles a GroEL subunit. However, the TRiC proteins contain an additional segment that acts as a built-in lid, so the complex encloses a polypeptide chain and mediates protein folding without the assistance of a GroES-like cochaperone. Like its bacterial counterpart, TRiC operates in an ATP-dependent fashion. Around 10% of eukaryotic proteins transiently interact with TRiC.
Chaperones Facilitate Protein Evolution
Chaperones may reduce the effects of a mutation in a protein that would otherwise preclude its proper folding. Subsequent mutations could then improve the protein’s folding efficiency and solubility, thereby onreducing its dependence on chaperones and increasing its abundance. Thus, chaperones increase the range of mutations that are subject to Darwinian selection.