Protein Tertiary and Quaternary Structure

Hierarchy of Protein Structural Organization

• Primary ($1^\circ$) Structure: Defined as a specific, linear sequence of amino acids linked by peptide bonds.

• Secondary ($2^\circ$) Structure: Refers to local folding patterns into repeating units, such as $\alpha$-helices and $\beta$-sheets, stabilized by hydrogen bonding.

• Tertiary ($3^\circ$) Structure: The comprehensive folding of a single polypeptide chain into its three-dimensional (3D) conformation.

• Quaternary ($4^\circ$) Structure: The association of multiple individual polypeptide chains (subunits) into a larger, functional protein complex.

Functional Classes: Fibrous vs. Globular Proteins

• Fibrous Proteins:
      * Characterized by extended structures.
      * Consist of repetitions of a single, uniform type of $2^\circ$ structure.
      * Biological function is primarily structural, providing support to cells and tissues.
      * Examples:
          * Keratin: Composed entirely of helices.
          * Silk Fibroin: Composed entirely of $\beta$-sheets.

• Globular Proteins:
      * Characterized by compact, spherical structures.
      * Involve specific packing interactions between various $2^\circ$ structural elements.
      * Carry out most of the chemical work within the cell.
      * Functions include molecular synthesis, transport, metabolism, and signaling.
      * Examples: Myoglobin, Neuraminidase, and Triosephosphate isomerase (TIM).

Representations of Tertiary Structure

• Using the protein Ubiquitin as a model, $3^\circ$ structures can be represented in four primary ways:
      * Cartoon: Highlights the backbones and secondary structures (helices and sheets).
      * Stick: Displays every atom and bond within the polypeptide.
      * Surface: Shows the overall physical boundary of the folded protein.
      * Surface with Electrostatic Potential: Maps the distribution of charges (positive and negative) across the protein's exterior.

Protein Domains: The Functional Units of Folding

• Definition: A domain is a distinct region of a compact, locally-folded $3^\circ$ structure within a larger protein.

• Characteristics:
      * Independent Folding: A domain is capable of folding into its stable structure independently of the rest of the protein.
      * Defined Function: Each domain possesses a specific function, such as DNA recognition, oligomerization, or cofactor binding.
      * Evolutionary Conservation: The same domain sequence and structure can be found across different proteins, where it maintains its general fold and function.

• Scale: While smaller proteins may consist of a single domain, larger proteins often contain two or more distinct domains (e.g., the Helix-loop-helix DNA binding domain).

Principles Governing Globular Protein Folding

• The Hydrophobic Effect:
      * Despite no specific pattern of hydrophobic or hydrophilic residues in the $1^\circ$ sequence, the folding process is driven by the sequestering of hydrophobic side chains.
      * Folding causes hydrophobic residues to pack tightly in the protein's interior (the "inside").
      * Hydrophilic residues are positioned on the protein's surface to interact with the aqueous environment (the "outside").

• Structural Twisting and Barrel Formation:
      * $\beta$-sheets are rarely flat; they typically twist or wrap into barrel structures (e.g., the $\beta$-barrel found in TIM and Neuraminidase).
      * This morphology is attributed to the inherent chirality of L-amino acids.

• Turns and Directional Changes:
      * Globular proteins must turn corners to transition between $\beta$-segments or $\alpha$-helices.
      * Turns often occur on the protein surface.
      * Proline is frequently involved due to its ability to make tight changes in direction and break $\alpha$-helices.
      * Glycine is often used in tight turns to prevent steric clashes with the carbonyl ($C=O$) group.
      * $\beta$-Turns: Classified as Type I or Type II, usually spanning four residues ($i, i+1, i+2, i+3$).
      * $\gamma$-Turns: A tighter turn frequently involving Proline at $i+1$ between residues $i$ and $i+2$.

• Loops and Disordered Regions:
      * Loops: Irregularly structured regions that connect defined $2^\circ$ elements.
      * Intrinsically Disordered Regions (IDRs): Segments of the protein that lack a fixed 3D structure.
      * These regions are often flexible to facilitate protein-protein or protein-ligand interactions and alter protein function (e.g., Cytochrome c, Tyrosine kinase).

Factors Stabilizing Tertiary Structure

• Noncovalent Interactions: Fundamental to all proteins for stabilizing $3^\circ$ structure.

• Disulfide Bonds (Covalent):
      * Provide additional stabilization in some proteins, particularly those exported from the cell into external environments.
      * The cellular interior is a reducing environment (unfavorable for disulfides), whereas external environments are often oxidizing (stabilizing for disulfides).
      * Examples: Ribonuclease, insulin, antibodies, and BPTI (Bovine Pancreatic Trypsin Inhibitor).

• Prosthetic Groups:
      * Small molecules (covalent or noncovalent) bound to the protein that are essential for function.
      * Holoprotein: The protein with its required prosthetic group bound.
      * Apoprotein: The protein without its prosthetic group.

Quaternary ($4^\circ$) Structure and Symmetry

• Definition: The association of multiple folded polypeptide chains to form specific multisubunit complexes.

• Stabilization: Complexes are held together by interactions between (not within) the folded polypeptides. These include:
      * Salt bridges
      * Hydrogen bonds
      * Van der Waals forces
      * The hydrophobic effect
      * Disulfide bridges

• Types of Subunit Composition:
      * Homotypic: Made up of a single type of subunit (all subunits are identical).
      * Heterotypic: Composed of different types of subunits (e.g., RNA polymerase II, TFIIA-RNA, TBP).

• Symmetry in $4^\circ$ Structure: Although individual polypeptides are asymmetric, their complexes often exhibit symmetry.
      * Example: Phosphofructokinase, a tetrameric enzyme, exhibits $D_2$ symmetry (viewed down three mutually perpendicular $C_2$ axes).
      * Example: The GroEL-GroES chaperone complex, which features a cis ring and a trans ring.