Protein Folding and Tertiary Structure Study Guide

Christian Anfinsen and Ribonuclease A (RNase A)

• Christian Anfinsen's Research: Anfinsen studied the activity and stability of Ribonuclease A (RNase A) to understand how proteins fold.
• Ribonuclease A (RNase A):
  • Source: Can be isolated in large quantities from cow pancreas.
  • Stability: Highly stable under various conditions; functions in cow stomachs at very low $pH$.
  • Assay for Activity: RNase A activity is measured using RNA solutions. RNA solutions are naturally viscous, and RNase A cleaves bonds within the RNA to make the solution less viscous.
  • Structural Features (Circa 1957):
    • Consists of a $124$ amino acid protein sequence.
    • Contains $4$ disulfide linkages ($S-S$ bonds).
    • The disulfide bonds provided a specific avenue for studying the partial folding and renaturation of the protein.

Protein Denaturation and Renaturation

• Chaotropes: Small molecules (such as urea or guanidinium chloride) commonly used to denature proteins by disrupting the hydrophobic core.
• Anfinsen’s Renaturation Experiments:
  • With $4$ disulfide bonds (formed from $8$ cysteine residues), there are $105$ possible combinations of disulfide pairings.
  • Statistically, only $1$ out of these $105$ combinations is the "right" native conformation.
• Disulfide Switching:
  • If a protein is denatured and the disulfides are reduced, the protein can become trapped in a "scrambled" (inactive) conformation if allowed to refold incorrectly.
  • A reducing environment allows for "disulfide switching," where bonds are broken and reformed until the protein reaches its native, thermodynamically stable state.
  • Renaturing with preformed disulfides also allows for the switching necessary to find the correct pairings.

Anfinsen’s Postulate and Fundamental Conclusions

• Conclusion 1: Proteins can fold spontaneously into their native conformation under physiological conditions, independent of the cellular environment (e.g., chaperones are not strictly required for the information of the fold, though they may assist in vivo).
• Conclusion 2: The functional, folded state of RNase A (the native state) must necessarily be the thermodynamically favored form.
  • This principle applies to all proteins.
  • Logic: If the functional form were not the thermodynamically favored form, proteins would spontaneously fold into a different, inactive form over time.

The Mechanism of Protein Folding

• Step-by-Step Folding Process:
  1. Structural Nucleation: Based on local structural preferences, small regions of the protein begin to form structures similar to (but not exactly) secondary structures.
  2. Hydrophobic Collapse: Local structures undergo a rapid collapse into a "molten globule" state, driven by the exclusion of water from hydrophobic side chains.
  3. Stabilization: Secondary structures are stabilized, internal side chains pack together tightly, and water is completely expelled from the protein core.

The Folding Funnel and Thermodynamics

• The Folding Funnel Concept: Proteins fold via a series of conformational changes that reduce their free energy ($G$) and entropy ($S$) until the native state (the global minimum) is reached.
• Characteristics:
  • There is no single structural element required as a universal folding intermediate.
  • There are many different paths a polypeptide chain can take to reach the bottom of the funnel.
  • Molten Globule: Represents a state of rapid hydrophobic collapse occurring early in the funnel.
• Entropy Paradox: While the second law of thermodynamics states entropy generally increases, the entropy value in the funnel is largest in the unfolded state and smallest in the folded state because the unfolded protein has many more possible conformations (higher configurational entropy).
• 3D-Folding Funnel: A more realistic energy landscape containing several potential local minima (traps) and thermodynamic barriers. Structures can be transiently trapped in these local minima (misfolded or intermediate states).

Protein Stability and Denaturation

• Marginal Stability:
  • Proteins are only marginally stable.
  • An average $100$-residue protein is stable by approximately $40\,kJ/mol$.
  • An average hydrogen bond provide approximately $20\,kJ/mol$ of stabilization.
  • Therefore, the loss of only two or three hydrogen bonds provides enough energy ($40\,kJ/mol$) to unfold a protein.
• Denaturation Defined: The state where a protein is unfolded or the secondary/tertiary structures are disrupted to create a "random coil."
• Melting Temperature ($T_m$): The temperature at which the transition between the folded structure and the unfolded/random coil structure occurs.
• Two-State Protein Folding: Folding is a cooperative process. When one part of the structure is disrupted, the interactions between it and the rest of the protein are lost, quickly destabilizing the entire structure. Transitions are abrupt, suggesting that folding intermediates are unstable and transient.
• Monitoring Denaturation: Techniques like Circular Dichroism (CD) measure secondary structure to monitor unfolding as a function of temperature or chaotrope concentration.

The Role of Water and the Hydrophobic Effect

• Solvent Driving Force: Water as a solvent provides the primary driving force for protein folding.
• Hydrophilic: "Water loving"; forms favorable interactions with water.
• Hydrophobic: "Water fearing/hating"; lacks favorable interactions with water.
• Amphipathic Secondary Structures:
  • Antiparallel $\beta$-sheet: If every other side chain is nonpolar and the rest are polar, the sheet usually sits at the protein surface. Nonpolar residues point toward the hydrophobic core, while polar residues interact with the aqueous solvent.
  • Amphipathic $\alpha$-helix: Hydrophobic residues often appear at the $i$, $i+3$, and $i+4$ positions to create one hydrophobic face and one hydrophilic face.
• Desolvation Cost:
  • In an unfolded protein, polar groups form hydrogen bonds with water.
  • Breaking these bonds to fold the protein requires energy (the desolvation cost).
  • Forming new hydrogen bonds within the protein interior compensates for this cost.
  • Unpaired Polar Groups: If a polar group is buried in the core without forming a hydrogen bond, it is energetically unfavorable and destabilizes the protein because the desolvation cost was not recovered.
• Entropy-Driven Folding: Protein folding is primarily driven by entropy (the hydrophobic effect), but stability is a fine balance between enthalpic and entropic forces.
  • Hydrophobic Effect: Drives the protein to a globular shape.
  • Hydrogen Bonds: Provide specificity, driving the specific type of globular shape formed by minimizing unpaired polar groups in the core.

Protein Misfolding and Diseases

• Prion Diseases:
  • Examples: Scrapie (sheep), Wasting Disease (deer), Mad Cow Disease (Bovine Spongiform Encephalopathy), and Creutzfeldt-Jakob disease (CJD, humans).
  • $PrP^C$: The normal "cellular prion protein"; not infectious. Residues $23$-$120$ have no detectable structure.
  • $PrP^{Sc}$: The "scrapie prion protein"; harmful and infectious. It has a high $\beta$-sheet content.
  • Mechanism: $PrP^{Sc}$ catalyzes the conversion of normal $PrP^C$ into the harmful $PrP^{Sc}$ form, leading to amyloid fibrils.
• Alzheimer’s Disease:
  • Characterized by $\beta$-amyloid plaques and neurofibrillary tangles.
  • Protein $A\beta$: Damage is thought to be caused by semi-aggregated precursors rather than the mature visible amyloid plaques.
  • Early-onset Alzheimer's: Linked to mutations that make these proteins less stable.
• Huntington’s Disease: Associated with polyQ (polyglutamine) sequences.

Fibrous Proteins

• General Characteristics: Repeated secondary structures packed together; usually insoluble in water; found in strings (hair, silk) or connective tissue (bones).
• Keratin:
  • Structure: Smallest assembly unit is a coiled-coil of $\alpha$-helices based on simple hydrophobic packing rules.
  • Locations: Major protein in fingernails, hair, animal wool, claws, quills, horns, hooves, and skin.
  • Stability: Disulfide bonds across helices provide quaternary structure stability.
• Silk (Soft Fibrous Protein):
  • Structure: Composed of silk fibroin, which consists of Glycite-Alanine (Gly-Ala) repeats in an extended $\beta$-sheet structure.
  • Mechanical Properties: Does not stretch easily because the $\beta$-strands are already fully extended. However, it is very flexible due to weak non-covalent interactions (hydrogen bonds between strands and Van der Waals interactions between sheets).
• Collagen (Complex Fibrous Protein):
  • Locations: Major protein in connective tissue (bone, skin, tendons, blood vessels, cartilage).
  • Primary Sequence: Follows a Gly-X-Y repeat, where the consensus is Gly-Pro-Hyp (Hydroxyproline).
  • Secondary Structure: Individual left-handed collagen helices.
  • Quaternary Structure: Three left-handed coils wrap around each other to form a right-handed triple helix. This counter-winding makes it resistant to compression (similar to a rope).
  • Post-translational Modifications:
    • Hydroxyproline (Hyp): Proline is converted to Hyp by the enzyme prolyl 4-hydroxylase. This residue changes the ring structure to favor helix formation.
    • Allysine: Modified lysine containing an aldehyde group.
    • Crosslinks: Covalent bonds form between the allysine aldehyde and a lysine amine, linking individual helices together to strengthen the fibril.

Collagen and Scurvy

• Enzymatic Mechanism:
  • Prolyl 4-hydroxylase requires $Fe^{2+}$ as a cofactor to convert Pro to Hyp.
  • Occasionally, $\alpha$-ketoglutarate reacts with the enzyme in the absence of Proline, oxidizing the iron to $Fe^{3+}$, which inactivates the enzyme.
• Vitamin C (Ascorbate): Acts as a reducing agent to reduce $Fe^{3+}$ back to $Fe^{2+}$, reactivating the enzyme.
• Scurvy: In the absence of Vitamin C, collagen cannot be processed correctly (lack of Hyp and crosslinks), leading to skin sores and tooth loss. This condition is reversible by consuming Vitamin C.
• Dietary Sources: Salad, citrus, peppers, tomatoes, and broccoli.
• Safety Note: Excessive Vitamin C consumption has an upper limit; too much can lead to kidney stones.

Questions & Discussion

• Keratin Fibrils: Hair splits easily along the fiber axis, but fingernails split across the nail. This implies that keratin fibrils in hair are oriented parallel to the growth axis, while in fingernails, they are likely layered or oriented in a way that resists longitudinal splitting.
• Polyproline Sequences: Polyproline can form a helical structure (Polyproline helix). Since Proline and Hydroxyproline drive the specific collagen structure, polyproline sequences are related to the formation of collagen-like structures, though collagen specifically requires the Glycine at every third position to allow the triple helix to pack tightly in the center.