Protein Folding and Tertiary Structure Study Guide

Historical Context of Protein Folding: Christian Anfinsen

• Christian Anfinsen conducted seminal research on the activity and stability of Ribonuclease A (RNase A).

• RNase A is a particularly useful model protein for the following reasons:

  • It can be isolated in large quantities from cow pancreas.

  • It is remarkably stable under various conditions, such as the low $pH$ environments found in cow stomachs.

  • Its activity is easily assayed; RNA solutions are inherently viscous, and RNase A cleaves bonds within RNA to make the solution less viscous.

  • The presence of several disulfide bonds provides a specific avenue for studying the partial folding and renaturation of the protein.

• Understanding of RNase A Structure (Circa 1957):

  • The protein consists of an primary sequence of $124$ amino acids.

  • It contains $4$ disulfide linkages.

  • Developing this understanding required many years of work in primary sequence analysis.

Chaotropes and Anfinsen’s Renaturation Experiments

• Chaotropes are small molecules commonly used to denature proteins by disrupting the hydrophobic core.

• In his renaturation experiments, Anfinsen noted that for a protein with $4$ cysteines (forming $4$ disulfide bonds), there are $105$ possible combinations of disulfide bonds.

• Statistically, only $1$ of these $105$ combinations is the "right" or native conformation.

• Disulfide Switching by Thiols:

  • Proteins can become trapped in a "scrambled" or misfolded conformation.

  • In a reducing environment, the protein undergoes slow refolding involving disulfide switching to find the native state.

  • Renaturing with preformed disulfides allows for this switching to occur until the most stable form is achieved.

Anfinsen’s Postulates and the Mechanistic Path of Folding

• Fundamental Conclusions (Anfinsen’s Postulate):

  1. Proteins can fold spontaneously into their native conformation under physiological conditions independent of the cellular environment.

  2. The functional, folded form of RNase A (and by extension, all proteins) must necessarily be the thermodynamically favored form.

  • If the functional form were not the thermodynamically favored form, proteins would spontaneously fold into inactive, non-functional forms.

• The Step-by-Step Process of Protein Folding:

  1. Structural Nucleation: Based on local structural preferences, small regions form structures similar to, but not precisely, secondary structures.

  2. Hydrophobic Collapse: Local structures undergo a collapse into a "molten globule" state.

  3. Stabilization: Secondary structures are stabilized, internal side chains pack together tightly, and water is expelled from the protein core.

The Folding Funnel and Thermodynamics

• The Folding Funnel Model:

  • Proteins fold via a series of conformational changes that reduce both free energy and entropy until the native state is reached.

  • There is no single structural element required as a universal folding intermediate.

  • Multiple paths exist to reach the bottom of the funnel (the native state).

  • The process involves a rapid hydrophobic collapse to the molten globule.

• Entropy in the Funnel:

  • The "entropy" value of the funnel is largest in the unfolded state because there are a vast number of possible disordered conformations.

  • Misfolded proteins represent local minima or "trap" points on the energy landscape.

• 3D-Folding Funnel:

  • A realistic energy landscape features several potential local minima and thermodynamic barriers.

  • Structures can be transiently trapped in these local minima before reaching the global minimum (native state).

Protein Stability and Denaturation

• Proteins are Marginally Stable:

  • An average $100$-residue protein is stable by approximately $40\,kJ/mol$.

  • Providing $40\,kJ/mol$ of energy (via heat or chemical disruption) is sufficient to unfold the protein.

  • The average hydrogen bond provides approximately $20\,kJ/mol$.

  • Mathematically, the loss of only two or three hydrogen bonds can be enough to unfold an entire protein.

• Denaturation Definitions:

  • Denatured proteins are those in which secondary and tertiary structures have been disrupted, resulting in a random coil.

  • The Melting Temperature ($T_m$) is the specific temperature at which the transition between the folded structure and the unfolded/random coil occurs.

• Two-State Protein Folding (Cooperative Folding):

  • Folding is cooperative; when one part of the structure is disrupted, the interactions maintaining the remaining structure are also weakened, leading to a rapid total destabilization.

  • Folding intermediates are generally unstable and transient.

  • Abrupt transitions observed in circular dichroism (which measures secondary structure) suggest this cooperative, two-state model.

The Role of Water and the Hydrophobic Effect

• Water as the Primary Driving Force:

  • Protein folding is primarily driven by entropy, specifically the entropy of water molecules.

  • Hydrophilic ("water-loving") groups form favorable interactions with water.

  • Hydrophobic ("water-fearing") groups do not make favorable interactions and drive folding to be sequestered from the solvent.

• Amphipathic Secondary Structures:

  • In antiparallel $\beta$-sheets, if every other side chain is nonpolar and the rest are polar, the sheet will likely reside at the protein surface. Nonpolar residues point toward the hydrophobic core, while polar residues interact with the aqueous solvent.

  • In an amphipathic $\alpha$-helix, hydrophobic residues typically occupy the $i$, $i+3$, and $i+4$ positions to create a nonpolar face.

• Desolvation Cost:

  • In an unfolded state, polar groups form hydrogen bonds with water.

  • Breaking these bonds to fold the protein requires an input of energy, known as the desolvation cost.

  • Forming new hydrogen bonds within the protein interior compensates for this cost.

  • Unpaired polar groups buried in the core are energetically unfavorable because they do not recover the desolvation cost.

• Summary of Folding Drivers:

  • Hydrophobic Effect (Entropy): Drives the protein to fold into a globular shape.

  • Hydrogen Bonds: Provide specificity rather than stability; they minimize unpaired polar groups in the core and determine the specific type of globular shape/secondary structure formed.

Protein Misfolding and Disease

• Diseases associated with misfolding include:

  • Alzheimer’s Disease: Characterized by $\beta$-amyloid ($A\beta$) plaque formation.

  • Huntington’s Disease: Associated with polyQ sequences.

  • Prion Diseases: Scrapie (sheep), Wasting Disease (deer), Mad Cow Disease (bovine spongiform encephalopathy), and Creutzfeldt-Jakob disease (CJD, humans).

• Prion Mechanism:

  • $PrP^C$ is the normal "cellular prion protein" and is not infectious.

  • $PrP^{Sc}$ is the "scrapie prion protein," which is infectious and harmful.

  • $PrP^{Sc}$ catalyzes the conversion of $PrP^C$ into more $PrP^{Sc}$.

  • The $PrP^{Sc}$ model structure contains significantly more $\beta$-sheet than $PrP^C$, eventually forming amyloid fibrils.

• Alzheimer’s Complexity:

  • Damage is thought to be caused by semi-aggregated protein precursors rather than the visible amyloid plaques themselves.

  • Mutations that destabilize involved proteins can lead to early-onset Alzheimer’s.

Fibrous Proteins: Keratin and Silk

• General Characteristics:

  • Usually insoluble in water.

  • Consist of repeated secondary structures packed together.

  • Found in strings (silk, hair) and connective tissues (bones, skin).

• Keratin:

  • The major protein in fingernails, hair, wool, claws, quills, horns, hooves, and skin.

  • Smallest assembly unit is a coiled-coil of $\alpha$-helices based on simple hydrophobic packing rules.

  • Quaternary structure is stabilized by disulfide bonds across helices.

• Silk:

  • Composed of silk fibroin, which consists essentially of Gly-Ala repeats in an extended $\beta$-sheet structure.

  • Does not stretch easily because the structure is already fully extended.

  • Very flexible due to weak non-covalent interactions between layers (hydrogen bonding between strands and Van der Waals interactions between sheets).

Fibrous Proteins: Collagen

• Structural Role:

  • Major protein in connective tissue: bone, skin, tendons, blood vessels, and cartilage.

• Sequence and Structure:

  • Primary sequence is a repeating triplet: Gly-X-Y, where the consensus sequence is Gly-Pro-Hyp (Hydroxyproline).

  • Proline and Hydroxyproline drive the structure.

  • Individual coils are left-handed helices.

  • Three left-handed coils wrap around each other to form a right-handed triple helix.

  • The counterwinding (like a rope) makes it highly resistant to compression.

• Post-translational Modifications:

  • Hydroxyproline: Modification of proline changes the preferred ring structure, aiding helix formation.

  • Allysine: Modified lysine containing an aldehyde group.

  • Covalent crosslinks form between the allysine aldehyde and a lysine amine, linking individual helices together to strengthen the fibril.

Collagen, Scurvy, and Vitamin C

• Biochemical Pathway:

  • The conversion of Proline to Hydroxyproline (Hyp) is performed by the enzyme prolyl 4-hydroxylase via oxidation-reduction.

  • In the catalysis, $\alpha$-ketoglutarate may react with the enzyme without Proline being present, oxidizing the catalytic $Fe^{2+}$ to $Fe^{3+}$, which inactivates the enzyme.

  • Vitamin C (ascorbate) is essential for the second reaction that reduces $Fe^{3+}$ back to $Fe^{2+}$, thereby reactivating the enzyme.

• Scurvy:

  • In the absence of Vitamin C, collagen cannot be processed correctly (lack of Hyp), leading to scurvy.

  • This is reversible by consuming Vitamin C (found in citrus, peppers, broccoli, etc.).

  • Excessive Vitamin C consumption has an upper limit and can lead to issues like kidney stones.

Important Concepts and Questions

• Question: Hair splits easily along the fiber axis, but fingernails tend to split across the nail rather than along it. What does this tell you about the direction of keratin fibrils in hair and fingernails? Why?

• Question: Polyproline sequences are known to form a helical structure. Do you think a polyproline sequence would form a collagen-like triple helix? Why?