The Problem of Folding and Protein Aggregation
At all stages of a protein's life—from synthesis on the ribosome and transport across membranes to oligomer assembly—intermediate, highly unstable conformations inevitably arise. In such states, hydrophobic residues, which are normally hidden deep inside the globule in a properly folded protein, become exposed on the surface.
Because the cell maintains a high concentration of various molecules, these exposed hydrophobic regions tend to immediately bind to one another. This leads to the aggregation (clumping) of poorly soluble proteins. Spontaneous renaturation under such conditions is severely hindered, which is why nature created special protective proteins—chaperones—to solve this problem.
Their basic tasks include:
- Ensuring optimal folding (formation of the native structure).
- Stabilizing the native conformation during protein function.
- Maintaining structure during disruptions in cellular homeostasis.
Classification of Molecular Chaperones
Chaperones are generally classified according to two main criteria: subunit molecular weight and the type of synthesis depending on the presence of stress factors.
By Subunit Molecular Weight:
- High-molecular-weight (60–110 kDa). These are entire families of related proteins. The best-studied representatives are the Hsp60, Hsp70, and Hsp90 classes. For example, the Hsp70 class includes proteins ranging from 66 to 78 kDa in mass.
- Low-molecular-weight (15–40 kDa).
By Stress Response:
- Constitutive. They have a high and constant (basal) level of synthesis, which does not depend on whether the cell is experiencing stress or not.
- Inducible (Heat Shock Proteins, Hsps). Under normal conditions, they are synthesized at low levels, but their production increases dramatically during stress. They got their name because they were first discovered during temperature stress that threatened proteins with denaturation.
Mechanism of Action of Hsp70 Chaperones
The Hsp70 class is highly conserved. These molecules are present in all cellular compartments: the nucleus, cytoplasm, mitochondria, and endoplasmic reticulum.
Structurally, Hsp70 proteins consist of a single polypeptide chain with a peptide-binding groove at the carboxy-terminal end. They recognize short segments 7 to 9 amino acids in length, rich in hydrophobic residues. In typical globular proteins, such sites occur approximately every 16 amino acids.
Role of Hsp70 in Protein Synthesis (Stage A): During translation on the ribosome, the N-terminus of a protein appears earlier than the C-terminus, but proper folding requires the entire chain. Hsp70 proteins bind to the polypeptide co-translationally (directly during synthesis). They cover dangerous hydrophobic regions with their groove, preventing premature aggregation before synthesis is fully complete.
Structure and Functions of Hsp60 Chaperones
Hsp60 chaperones come into play after chain synthesis is complete (Stage B). Their main task is to ensure the folding of high-molecular-weight proteins with complex conformations (e.g., those with domain structures).
Unlike Hsp70, Hsp60 proteins function as a huge oligomeric complex consisting of 14 subunits. They form a cylindrical cavity made of two stacked rings (7 subunits in each).
Each subunit has three domains:
- Apical (terminal). Faces the inside of the cavity, enriched with hydrophobic residues for the initial binding of the unfolded protein.
- Intermediate.
- Equatorial (at the base). ATP hydrolysis takes place here.
A protein with hydrophobic markers enters the isolated cavity of this complex. A specific environment is created inside, where continuous testing of possible spatial structures occurs until the single most energetically favorable form is found. This process requires significant energy expenditure in the form of ATP.
Additional Functions of Chaperones
Protection against denaturation and folding of new chains are not the only tasks of these proteins. Chaperones are also necessary for the cell to:
- Correctly assemble complex oligomeric proteins from multiple subunits.
- Transport proteins across cell and organelle membranes.
- Regulate the functional activity of protein complexes.
- Recognize irreversibly denatured proteins and target them for degradation in lysosomes.