Premed · Premed · Biochemistry
Lecture 5: Protein Folding and Misfolding Diseases
Biochemistry
Learning Objectives
By the end of this lecture, students will be able to:
- Explain Anfinsen's experiment and the thermodynamic hypothesis of protein folding
- Describe the role of molecular chaperones in assisted protein folding
- Explain the concept of the protein folding energy landscape
- Describe the mechanisms of protein misfolding and aggregation
- Identify the major misfolding diseases and their molecular basis
- Explain the role of the ubiquitin-proteasome system and autophagy in protein quality control
Lecture Content
I. The Protein Folding Problem
A central question in biochemistry is how a linear amino acid sequence folds into a specific three-dimensional structure. Levinthal's paradox illustrates the magnitude of this challenge: if each residue could adopt just 3 conformations, a 100-residue protein would have approximately 3^100 (roughly 5 x 10^47) possible conformations. A random search of all these conformations would take longer than the age of the universe, yet proteins fold in milliseconds to seconds. This paradox demonstrates that folding must follow a directed pathway rather than a random search.
II. Anfinsen's Experiment (Thermodynamic Hypothesis)
Christian Anfinsen's classic experiment with ribonuclease A (RNase A), which earned him the 1973 Nobel Prize, provided a foundational insight into protein folding. He denatured RNase A with urea (to unfold the protein) and beta-mercaptoethanol (to reduce disulfide bonds), completely abolishing enzyme activity. When he removed both denaturants by dialysis, the protein refolded spontaneously and regained full activity, with all four disulfide bonds re-forming correctly out of 105 possible pairings.
This experiment established the thermodynamic hypothesis: the native structure of a protein is the thermodynamically most stable state under physiological conditions. In other words, the amino acid sequence contains all the information needed for correct folding, and the native state represents the global free energy minimum.
III. The Folding Energy Landscape
Protein folding is best understood as movement on a funnel-shaped energy landscape. The width of the funnel represents conformational entropy (the number of possible conformations), while the depth represents enthalpy (stabilizing interactions). Unfolded states sit at the top of the funnel with high entropy and high energy, and the native state resides at the bottom with low entropy and low energy. Folding is driven by a decrease in free energy (delta-G less than 0), though the margin is surprisingly slim: native proteins are only 20 to 60 kJ/mol more stable than the unfolded state.
Along the folding pathway, proteins can pass through intermediates such as the molten globule, a compact state with native-like secondary structure but fluctuating tertiary structure. Local energy minima on the landscape can trap misfolded intermediates. The key driving forces are hydrophobic collapse (the rapid burial of nonpolar residues), the formation of secondary structure elements, and specific side chain packing in the final stages.
<image>A protein folding energy funnel diagram. The funnel shape has a wide top representing the unfolded ensemble (high entropy, many conformations) and narrows to a single point at the bottom representing the native state (low energy, single conformation). The vertical axis is free energy and the horizontal axes represent conformational coordinates. Intermediate states are shown as local minima on the funnel walls, including a molten globule intermediate. Misfolded aggregates are shown as a separate, deep energy well off to the side of the funnel, illustrating that aggregation can be thermodynamically favorable but kinetically avoided.</image>
IV. Molecular Chaperones
Many proteins require assistance to fold correctly in the crowded cellular environment. Chaperones do not provide folding information; instead, they prevent misfolding and aggregation.
Hsp70 System (Heat Shock Protein 70)
Hsp70 binds to exposed hydrophobic segments of nascent or unfolded polypeptides through an ATP-dependent binding and release cycle. In its ATP-bound form, Hsp70 has low affinity and binds and releases substrates rapidly (lid open). ATP hydrolysis, stimulated by the Hsp40 co-chaperone, closes the lid and increases binding affinity. A nucleotide exchange factor then replaces ADP with ATP, opening the lid and releasing the substrate. This cycle prevents premature aggregation and gives the polypeptide time to fold correctly. Hsp70 also participates in protein translocation across membranes and the disassembly of clathrin coats.
Chaperonin System (Hsp60/GroEL-GroES in bacteria)
The chaperonin system is a large barrel-shaped complex. GroEL consists of two stacked heptameric rings forming a central cavity, and GroES is a single heptameric ring that caps the GroEL barrel. A misfolded protein enters the GroEL cavity, which has a hydrophobic interior. Upon ATP binding, GroES caps the cavity, and the interior becomes hydrophilic, providing a protected environment for folding. After approximately 10 seconds (the time required for ATP hydrolysis), GroES dissociates and the protein is released. If the protein is not properly folded, the cycle repeats. The eukaryotic equivalent of this system is the TRiC/CCT chaperonin.
Hsp90
Hsp90 is involved in the maturation of signaling proteins such as steroid receptors and kinases. It is currently a drug target, with Hsp90 inhibitors being developed as anticancer agents.
V. Protein Quality Control
The Ubiquitin-Proteasome System
Terminally misfolded proteins are targeted for degradation through the ubiquitin-proteasome system. Ubiquitin is a small 76-amino acid protein that is covalently attached to lysine residues on target proteins through the sequential action of three enzymes: E1 (activating enzyme), E2 (conjugating enzyme), and E3 (ligase, which provides substrate specificity). A polyubiquitin chain of at least 4 ubiquitins linked via Lys48 signals the protein for degradation. The 26S proteasome is a large barrel-shaped protease complex consisting of a 20S core with four stacked rings containing proteolytic active sites and 19S regulatory caps that recognize polyubiquitin chains, unfold the substrate, and thread it into the 20S core. The protein is cleaved into short peptides of 6 to 10 residues. This process requires ATP.
Autophagy
Autophagy is a bulk degradation pathway for damaged organelles and large protein aggregates. In macroautophagy, a double-membrane vesicle called an autophagosome engulfs cargo and fuses with a lysosome for degradation. This pathway is important for clearing aggregates that cannot be handled by the proteasome.
VI. Protein Misfolding Diseases
When protein quality control fails, misfolded proteins can accumulate and cause disease.
Amyloid Diseases
Amyloid diseases are characterized by the accumulation of insoluble protein aggregates with cross-beta structure known as amyloid fibrils. These fibrils have beta-strands oriented perpendicular to the fibril axis, stabilized by extensive hydrogen bonding. They are extremely stable and resistant to proteolysis. Diagnostically, amyloid fibrils stain with Congo red dye and show apple-green birefringence under polarized light. They also bind thioflavin T, which can be detected by fluorescence.
Specific Misfolding Diseases
Alzheimer disease is characterized by extracellular amyloid-beta plaques, formed from cleavage of amyloid precursor protein by beta-secretase and gamma-secretase, and by intracellular neurofibrillary tangles of hyperphosphorylated tau protein. The disease causes progressive neurodegeneration and dementia.
Parkinson disease features intracellular Lewy bodies composed of aggregated alpha-synuclein and involves the loss of dopaminergic neurons in the substantia nigra.
Huntington disease results from an expansion of CAG repeats in the huntingtin gene, producing a polyglutamine expansion. The mutant huntingtin protein aggregates in neuronal nuclei. The disease follows autosomal dominant inheritance and is classified as a trinucleotide repeat disorder.
Prion diseases (transmissible spongiform encephalopathies) involve the conversion of normal cellular prion protein (PrPc), which is predominantly alpha-helical, into the misfolded scrapie form (PrPSc), which is predominantly beta-sheet. PrPSc acts as a template, converting PrPc to PrPSc in a self-propagating manner -- making it an infectious protein that requires no nucleic acid for transmission. Examples include Creutzfeldt-Jakob disease, kuru, and bovine spongiform encephalopathy (mad cow disease).
Systemic amyloidoses include AL amyloidosis (from immunoglobulin light chain fragments), AA amyloidosis (from serum amyloid A protein in chronic inflammation), and transthyretin amyloidosis (from misfolded transthyretin deposits in the heart and nerves).
<image>A figure comparing normal protein folding with misfolding pathways. Panel A: Normal folding pathway from unfolded protein through intermediates to native state, assisted by chaperones. Panel B: Misfolding pathway showing how partially folded intermediates can form oligomers, protofibrils, and mature amyloid fibrils with cross-beta structure. An inset shows the cross-beta structure with beta-strands running perpendicular to the fibril axis. Panel C: Microscopy images of amyloid plaques stained with Congo red showing apple-green birefringence under polarized light. Panel D: The prion conversion mechanism showing PrPc (alpha-helical) being converted to PrPSc (beta-sheet) through a templating mechanism.</image>
VII. Protein Denaturation
Denaturation is the loss of native structure and function without breaking peptide bonds. Various agents can cause denaturation. Heat disrupts non-covalent interactions, and proteins have characteristic melting temperatures (Tm). Extreme pH alters charges on ionizable groups, disrupting salt bridges and hydrogen bonds. Urea and guanidinium chloride disrupt hydrophobic interactions and hydrogen bonds. Detergents such as SDS disrupt hydrophobic interactions. Reducing agents like beta-mercaptoethanol and DTT break disulfide bonds. Heavy metals such as mercury and lead react with sulfhydryl groups.
Denaturation can be reversible or irreversible depending on conditions. Small, single-domain proteins often refold spontaneously, as demonstrated in Anfinsen's experiment, while large, multi-domain proteins may require chaperones or may aggregate irreversibly.
<image>A diagram illustrating Anfinsen's ribonuclease A refolding experiment. Step 1: Native RNase A with four disulfide bonds shown in a ribbon diagram with enzymatic activity indicated. Step 2: Addition of 8M urea and beta-mercaptoethanol results in a fully unfolded, reduced polypeptide with no activity. Step 3: Removal of denaturants by dialysis leads to spontaneous refolding and re-formation of the correct disulfide bonds, restoring full activity. A side pathway shows that if beta-mercaptoethanol is removed first (in the presence of urea), random disulfide bonds form, yielding a scrambled, inactive protein that can be rescued by adding trace beta-mercaptoethanol.</image>
