The Three-Dimensional Structure of Proteins

Chapter 6: The Three-Dimensional Structure of Proteins

Overview

This chapter explores the hierarchical organization of protein structure, the principles governing protein folding, and the structural and functional diversity of proteins. Understanding these concepts is fundamental to biochemistry, as protein structure determines biological function.

Secondary Structure: Regular Ways to Fold the Polypeptide Chain

Pauling’s Rules for Secondary Structure

In the early 1950s, Linus Pauling and collaborators established several principles that secondary protein structures must obey:

• Bond Angles and Lengths: Should be similar to those found in free amino acids.

• Van der Waals Radii: No atoms should approach each other more closely than allowed by their van der Waals radii.

• Planarity of the Amide Group: The peptide bond is planar due to partial double-bond character, restricting rotation.

• Stabilization by Noncovalent Bonds: Particularly hydrogen bonds, which stabilize folding processes and products.

Common Secondary Structure Elements

• α-Helix: Side chains radiate outward from the helix axis; hydrogen bonds are nearly parallel to the helix axis. Often displays distinct hydrophilic and hydrophobic faces.

• β-Sheet: Stabilized by interchain hydrogen bonds; side chains alternate above and below the plane of the sheet. Strands can be parallel (N→C in the same direction) or antiparallel (N→C in opposite directions).

• 310-Helix: Meets Pauling’s criteria but is less common in proteins.

Rotation Around Bonds in a Polypeptide Backbone

• Rotation is restricted around the peptide bond due to its partial double-bond character.

• Rotation is allowed around the N–Cα (φ) and Cα–C (ψ) bonds, but steric hindrance limits the possible angles.

• These angles are described as φ (phi) and ψ (psi).

Ramachandran Plots

Ramachandran plots show the sterically allowed φ and ψ angles for polypeptides. White areas correspond to allowed conformations, while shaded areas indicate steric clashes. The allowed regions are smaller for amino acids with larger R groups.

Levels of Protein Structure

• Primary (1°) Structure: The linear sequence of amino acids in a polypeptide chain.

• Secondary (2°) Structure: Local regions of regular folding, such as α-helices and β-sheets.

• Tertiary (3°) Structure: The overall three-dimensional arrangement of secondary structural elements.

• Quaternary (4°) Structure: The spatial arrangement of multiple polypeptide chains (subunits) in a multisubunit complex.

Fibrous Proteins: Structural Materials of Cells and Tissues

Characteristics and Examples

• Fibrous Proteins: Elongated molecules with well-defined secondary structures, serving structural roles in cells and tissues.

• Examples:

  • Keratin: Found in hair, nails, feathers, and intermediate filaments.

  • Fibroin: Main protein in silk cocoons, composed of extensive β-sheets.

  • Collagen: Abundant connective tissue protein, forms a triple-stranded helix and is the matrix for bone mineralization.

Collagen Structure and Function

• Collagen has a repeating Gly-X-Y tripeptide motif, where X is often Proline and Y is often Hydroxyproline or Hydroxylysine.

• Triple-stranded left-handed helix stabilized by crosslinking and glycosylation.

• Vitamin C is required for proline hydroxylation; deficiency leads to collagen degeneration (scurvy).

Globular Proteins: Tertiary Structure and Functional Diversity

General Features

• Globular proteins have compact, complex tertiary structures with varying amounts of α-helix, β-sheet, and loop regions.

• Often composed of domains (~200 amino acids) that can fold and function independently.

• Domains often have specific functions (e.g., DNA binding, oligomerization, cofactor binding).

Classification of Protein Structure

• Proteins can be classified as mainly α, mainly β, α/β, or few secondary structures.

• Not all regions are regular; some are termed "random coil" or "irregularly structured regions."

Common Features of Folded Globular Proteins

• Nonpolar (hydrophobic) residues are typically buried in the interior, while hydrophilic residues are exposed to the solvent.

• β-sheets are often twisted or form barrel structures.

• Polypeptide chains can turn corners via β-turns or γ-turns.

Factors Determining Secondary and Tertiary Structure

Information for Protein Folding

• The amino acid sequence contains all the information necessary for folding into the native structure (Anfinsen’s experiment with ribonuclease A).

• Protein folding is a thermodynamically favorable process under physiological conditions.

Thermodynamics, Folding, and Stability

• Favorable Interactions: Intramolecular enthalpic interactions (ionic bonds, hydrogen bonds, van der Waals interactions).

• Unfavorable Entropy Loss: Folding reduces conformational entropy.

• Hydrophobic Effect: Burying hydrophobic groups increases solvent entropy by releasing ordered water molecules.

• Disulfide Bonds: Covalent S–S bonds increase stability by reducing the number of possible unfolded conformations.

• Binding of Ions or Prosthetic Groups: Can further stabilize protein structure.

Dynamics of Globular Protein Structure

Kinetics and Pathways of Protein Folding

• Levinthal’s paradox: Proteins fold rapidly despite the astronomical number of possible conformations.

• Folding involves intermediate states, such as the "molten globule" state, with native-like secondary structure but incomplete tertiary structure.

• Folding energy landscapes are often depicted as funnels, with the native state at the bottom.

Role of Chaperones

• Chaperones are proteins that assist in the proper folding of other proteins, especially under stress conditions.

• They prevent aggregation and misfolding, often by providing a protected environment for folding (e.g., GroEL/GroES system in Escherichia coli).

Quaternary Structure of Proteins

Symmetry in Quaternary Structures

• Multisubunit proteins can exhibit various symmetries, such as helical, cyclic (Cn), or dihedral (Dn) symmetry.

• Examples include actin filaments (helical symmetry), transthyretin (C2 symmetry), and phosphofructokinase (D3 symmetry).

• Heterotypic protein-protein interactions (between different proteins) are determined by complementary surfaces.

Tools of Biochemistry

Determining Molecular Masses and Subunit Composition

• Size Exclusion Chromatography: Separates proteins based on size by elution through a resin-filled column.

• Analytical Ultracentrifugation: Measures sedimentation equilibrium to determine molecular mass.

• Mass Spectrometry: Identifies mass-to-charge ratios of protein components.

• SDS-PAGE: Polyacrylamide gel electrophoresis under denaturing conditions estimates subunit molecular masses by comparison to reference proteins.

Summary Table: Levels of Protein Structure

Level	Description	Stabilizing Forces
Primary	Linear sequence of amino acids	Peptide bonds
Secondary	Local folding (α-helix, β-sheet)	Hydrogen bonds
Tertiary	3D arrangement of secondary structures	Hydrophobic interactions, ionic bonds, hydrogen bonds, disulfide bonds
Quaternary	Assembly of multiple polypeptide chains	Noncovalent interactions, sometimes disulfide bonds

Key Equations

• Ramachandran Angles: φ (phi) and ψ (psi) define the rotation around the N–Cα and Cα–C bonds, respectively.

• Hydrophobic Effect (Entropy): $\Delta S_{solvent} > 0$ when hydrophobic groups are buried, increasing overall entropy.

• Gibbs Free Energy of Folding: $\Delta G = \Delta H - T\Delta S$ Where $\Delta G$ is the change in free energy, $\Delta H$ is the change in enthalpy, $T$ is temperature, and $\Delta S$ is the change in entropy.