Protein Structure and Folding Review

Functional Implications of Protein Structure

• Proteins are responsible for performing the vast majority of biological work within a cell.

• The specific function of a protein is dictated entirely by its structure or shape. Therefore, a protein's function depends on its structure.

• Proteins exhibit diverse morphologies tailored to their roles:

  • Collagen: Characterized by a highly linear, elongated shape.

  • Insulin: A notably small protein relative to other cellular machinery.

  • Calmodulin: Features a "dumbbell" shape consisting of one globular domain connected to another globular domain via a linear region.

The Peptide Bond and Primary Structure

• Primary Structure: Refers to the linear sequence of amino acids linked together in a polypeptide chain.

• Peptide Bonds: Strong covalent bonds used to link individual amino acids together. There are three crucial characteristics of the peptide bond:

  1. Strength: It is a strong covalent bond, ensuring the amino acid chain does not spontaneously come apart.

  2. Structural Byproducts: Every peptide bond leaves behind a carbonyl oxygen ($O$) and an amide hydrogen ($H$). These atoms are essential for the formation of hydrogen bonds.

  3. Planarity/Rigidity: The bond is planar and rigid, preventing rotation specifically at the site of the bond itself.

• Termini:

  • N-terminus (Amino Terminus): The end of the peptide backbone with an exposed amino group.

  • C-terminus (Carboxy Terminus): The end of the peptide backbone with an exposed carboxyl group.

• R-Group Orientation: Side chains (R-groups) project off the peptide backbone. While often simplified as all pointing in the same direction, in reality, their orientation is limited by physical constraints:

  • Steric Hindrance: Two bulky side chains located next to each other may not have sufficient room, forcing one to move.

  • Electrostatic Repulsion: Two side chains with the same charge (e.g., both positive) will repel each other, influencing their orientation.

Secondary Structure: Alpha Helices and Beta Sheets

• Secondary structure refers to localized folding within specific regions of a protein. Approximately $60\%$ of a protein's structure is composed of these two most common types.

• Alpha Helix: A spiral staircase-like structure.

  • Bonding Pattern: Hydrogen bonds form between every first ($i$) and fourth ($i+4$) amino acid (e.g., residue $1$ to residue $4$, residue $2$ to residue $5$).

  • Mathematics of Rotation: It takes $3.6$ amino acids to complete a full $360^∘$ turn, yet the hydrogen bonding occurs every $4$ residues. This slight discrepancy means residues do not line up perfectly vertically.

  • Internal Stabilization: Hydrogen bonds are buried on the inside of the helix. While these bonds are hydrophilic, water is excluded from the interior, maintaining the structure. The R-groups point outward in all directions.

  • Prohibited Amino Acids: Proline (which kinks the backbone) and Glycine (with a side chain too small to stabilize the turn) are rarely found in alpha helices.

• Beta Sheets: Formed when segments of the peptide backbone lie parallel to one another.

  • Pleated Sheets: These are called pleated because the adjacent strands are not in the same horizontal plane. One segment may be in an elevated plane relative to the next.

  • Antiparallel Beta Sheets: The orientation of the strands is opposite (e.g., the N-terminus of one strand is next to the C-terminus of the adjacent strand). This requires only a small amount of random coil to connect the strands.

  • Parallel Beta Sheets: The strands have the same orientation. This requires a long segment of random coil to loop back and align the next strand in the same direction.

• Random Coil: Non-structured or disordered regions that connect alpha helices and beta sheets. Despite being "random," these regions are functionally significant, often serving as sites for phosphorylation or interaction.

Motifs and the Coiled-Coil

• Motif: A specific combination or association of secondary structures (e.g., an alpha helix associated with a beta sheet).

• Coiled-Coil Motif: A highly stable structure formed by two alpha helices twisting around each other.

  • Mechanism: In an amphipathic alpha helix, one side is polar and the other is nonpolar. Because turns occur every $3.6$ residues but nonpolar residues appear every $4$ residues, the nonpolar residues form a "stripe" that wraps around the helix like the stripe on a candy cane.

  • Interaction: Hydrophobic side chains on one helix interact with the hydrophobic stripe on another. This twisting provides significant structural strength and stability.

  • Side Chain Composition: These stripes usually consist of aliphatic (non-aromatic) hydrophobic side chains. Aromatic residues like Tyrosine, Phenylalanine, and Tryptophan are typically excluded.

Tertiary and Quaternary Structure

• Tertiary Structure: The full three-dimensional folding of a single polypeptide chain.

• Domains: Independent segments of a polypeptide chain that fold into compact, stable structures. Different domains often have unique functions:

  • Catalytic Domain: The part of an enzyme that catalyzes a reaction.

  • Substrate Binding Domain: The part responsible for binding the substrate.

  • SH2 and SH3 Domains: Common domains involved in cell signaling pathways.

• Quaternary Structure: The assembly of multiple separate polypeptide chains into a functional complex.

  • Homodimer: Two copies of the same protein.

  • Heterodimer: Two different proteins working together.

  • Tetramer: Four protein subunits.

  • Multimers: Large assemblies, such as the pyruvate transporter traversing the mitochondrial membrane, which is a $60$-mer (60 copies of the same protein).

• Macromolecular Machines: Large complexes of proteins (and sometimes RNA) that work together to perform tasks. The Ribosome is a classic example, consisting of ribosomal RNA and numerous proteins acting as a machine.

Molecular Affinity and On/Off Rates

• Affinity: The strength of the association between two molecules, defined as the ratio of the On-rate to the Off-rate.

  • On-rate: The rate at which molecules associate via random bumping into one another.

  • Off-rate: The rate at which molecules dissociate. This is determined by the number of non-covalent bonds formed.

• Mechanics of Binding: Interaction is not based on distant attraction but on random collisions. If the shapes and side chains (positive/negative charges, polar/nonpolar groups) allow for many non-covalent bonds (Hydrogen bonds, Van der Waals, electrostatic), the Off-rate will be low, resulting in high affinity.

The Biophysics of Folding and Denaturation

• Rotation around the Alpha Carbon: The peptide bond is rigid, but the bonds on either side of the alpha carbon can rotate. These are defined by two angles:

  • Phi ($\phi$): The angle between the nitrogen and the alpha carbon.

  • Psi ($\psi$): The angle between the carbonyl carbon and the alpha carbon.

• Energetically Favored Orientations: Plotting $\phi$ and $\psi$ angles reveals clusters of favored combinations:

  • Alpha Helices: Favored near angles of $-57$ and $-47$.

  • Beta Sheets: Favored near angles of $-125$ and $+25$.

• Oil Drop Model: In aqueous environments, proteins fold to bury nonpolar (hydrophobic) side chains inside the core, while polar (hydrophilic) side chains remain on the surface.

• Denaturation Agents: Chemicals or conditions that unfold proteins by disrupting non-covalent bonds:

  • Heat: Increases molecular motion, breaking non-covalent bonds.

  • pH Changes: Disrupts electrostatic attractions by changing the charge on acidic and basic side chains.

  • Urea: A small molecule with high hydrogen-bonding potential that competes with the protein's internal H-bonds.

  • $\beta$-mercaptoethanol: A reducing agent (antioxidant) required to break covalent disulfide bonds between Cysteine residues.

• Spontaneous Renaturation: If denaturing agents are removed, proteins will often refold spontaneously into their original native structure. This proves that the primary amino acid sequence contains all the information necessary for correct folding. Folding moves the protein from a high-energy, high-entropy (disordered) state to a low-energy, low-entropy (ordered) state.

Questions & Discussion

• Question (Student): Last time, you mentioned something about the arbors not lining up perfectly, making a full turn every $3.6$ but attaching every $4$. Why is that significant?

• Response: This discrepancy is what creates the angled hydrophobic "candy cane" stripe on alpha helices, which is necessary for creating motifs like the coiled-coil when two helices twist together.

• Question (Student): Can we get our textbook yet?

• Response: There are ongoing technical issues with access codes. The name of the textbook is visible, but the content is currently locked. The faculty is troubleshooting this with the provider.

• Question (Student): Could you define on-rate and off-rate?

• Response: The on-rate is the rate at which proteins or molecules associate. The off-rate is the rate at which they come apart. These rates are determined by how many non-covalent bonds (governed by shape and side chains) can form between the molecules.

• Discussion on Conservative Substitutions: A conservative substitution is a mutation where one amino acid is replaced by another with similar properties (e.g., Lysine to Arginine, or one nonpolar residue for another). Non-conservative substitutions (e.g., Glycine for Tyrosine) cause more significant structural and functional changes.

• Announcement: A quiz regarding nonpolar amino acids is scheduled for Tuesday.", "title": "Protein Structure, Motifs, and Folding Dynamics"}