Protein folding is the process by which a linear polypeptide chain adopts its native three-dimensional structure. While the amino acid sequence contains all the information needed for folding, as demonstrated by Anfinsen’s classic experiments with ribonuclease, many proteins require molecular chaperones to fold efficiently in the crowded cellular environment.
The Folding Problem
The number of possible conformations for a polypeptide chain is astronomically large. A protein of 100 amino acids could theoretically adopt an enormous number of conformations, yet folding occurs in milliseconds to seconds. This paradox, known as Levinthal’s paradox, shows that folding is not a random search but follows specific pathways. Folding is guided by hydrophobic collapse, hydrogen bonding, and the formation of secondary structural elements that restrict the conformational search.
The Energy Landscape
Protein folding is described by the energy landscape or folding funnel concept. The funnel-shaped landscape has many high-energy unfolded conformations at the top and a single low-energy native state at the bottom. The funnel is not smooth but contains local energy minima where partially folded intermediates can accumulate. Some proteins fold through well-defined intermediates, while others fold by more gradual compaction. Rugged landscapes with deep kinetic traps can lead to misfolding and aggregation.
Hydrophobic Collapse
The driving force for protein folding is the hydrophobic effect. Nonpolar side chains cluster together in the protein interior, minimizing their exposure to water. This hydrophobic collapse is thermodynamically favorable because it releases water molecules from the ordered solvation shells around hydrophobic surfaces, increasing entropy. The collapsed globule then undergoes structural rearrangements to form the native structure, with secondary and tertiary structure forming concurrently.
Molecular Chaperones
Molecular chaperones are proteins that assist folding without becoming part of the final structure. They prevent aggregation by binding exposed hydrophobic surfaces of unfolded or partially folded proteins. Chaperones do not contain steric information for folding but prevent inappropriate interactions that lead to misfolding.
Hsp70 Chaperone System
The Hsp70 family includes constitutively expressed Hsc70 and stress-induced Hsp70. Hsp70 recognizes exposed hydrophobic patches on unfolded proteins through its C-terminal substrate-binding domain. Binding and release are regulated by ATP hydrolysis in the N-terminal nucleotide-binding domain. J proteins, such as Hsp40, stimulate ATP hydrolysis and deliver substrates to Hsp70. Nucleotide exchange factors such as Bag-1 and HspBP1 facilitate ADP release and rebinding of ATP.
Chaperonins
Chaperonins are large barrel-shaped complexes that provide an isolated chamber for protein folding. Group I chaperonins in bacteria include GroEL and its cofactor GroES. GroEL forms a stacked double ring of seven subunits each, creating a central cavity. Unfolded protein binds in the cavity, GroES caps the chamber, and ATP hydrolysis induces conformational changes that release the protein into the enclosed space for folding. Group II chaperonins in eukaryotes, called TRiC or CCT, function without a separate cofactor and assist the folding of cytoskeletal proteins such as actin and tubulin.
Protein Disulfide Isomerase
Protein disulfide isomerase catalyzes the formation and rearrangement of disulfide bonds in the endoplasmic reticulum. It contains thioredoxin-like domains with active site cysteine residues that form mixed disulfides with substrate proteins. PDI can introduce, reduce, or isomerize disulfide bonds, ensuring that the correct pairing of cysteine residues is achieved as the protein folds. The redox state of the ER is maintained by the glutathione buffer, with an oxidized environment that favors disulfide formation.
Peptidyl-Prolyl Isomerases
Peptidyl-prolyl isomerases accelerate the cis-trans isomerization of proline peptide bonds, which is intrinsically slow and can be rate-limiting for protein folding. There are three families: cyclophilins, FK506-binding proteins, and parvulins. Cyclophilins are targets of the immunosuppressant cyclosporine A. PPIs are present in all cellular compartments and assist folding of proteins containing proline residues. PPI activity is particularly important for collagen folding, where glycine-proline-hydroxyproline repeats are abundant.
Misfolding and Disease
Protein misfolding and aggregation are associated with many diseases. Alzheimer disease involves the aggregation of amyloid-beta peptides into amyloid plaques and tau protein into neurofibrillary tangles. Parkinson disease features alpha-synuclein aggregation into Lewy bodies. Huntington disease results from polyglutamine expansion in huntingtin protein. Prion diseases involve the conversion of the normal prion protein into an abnormal, aggregation-prone conformation that is infectious. In all these diseases, the balance between protein folding, chaperone assistance, and degradation is disrupted.
