The Three-Dimensional Structure of Proteins

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Chapter 6: The Three-Dimensional Structure of Proteins

Introduction

This chapter explores the structural organization of proteins, focusing on the principles that govern their three-dimensional folding. Understanding protein structure is fundamental in biochemistry, as it underlies protein function and interactions in biological systems.

Secondary Structure: Regular Ways to Fold the Polypeptide Chain

Definition and Importance

Secondary structure refers to local, regularly repeating structures within a polypeptide chain, stabilized primarily by hydrogen bonds.
Common secondary structures include the α-helix and β-sheet.

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, contributing to protein folding and function.

β-Sheet

Stabilized by interchain hydrogen bonds.
Side chains alternate above and below the plane of the sheet.
Strands can be parallel (N→C direction the same) or antiparallel (N→C direction opposite).

Other Secondary Structures

310 helix: A less common helical structure with different hydrogen bonding patterns.
Turns and Loops: Connect elements of secondary structure and allow the polypeptide chain to reverse direction.

Three-Dimensional Folding of the Protein Myoglobin

Myoglobin Structure

Myoglobin is a globular protein composed almost entirely of α-helices.
Its structure demonstrates how secondary structure elements fold into a compact, functional three-dimensional form.
Contains a bound heme group essential for oxygen binding.

Rotation Around Bonds in a Polypeptide Backbone

Backbone Flexibility and Constraints

Rotation in the polypeptide backbone is possible around the N–Cα (φ, phi) and Cα–C (ψ, psi) bonds.
The peptide bond (C–N) has partial double-bond character, restricting rotation and keeping atoms in a planar arrangement (amide plane).
Steric hindrance limits the range of allowed φ and ψ angles, influencing possible secondary structures.

Levels of Protein Structure

Hierarchical Organization

Primary (1°) structure: The linear sequence of amino acids in a polypeptide chain.
Secondary (2°) structure: Local regions of regular folding (e.g., α-helix, β-sheet).
Tertiary (3°) structure: The overall three-dimensional arrangement of all atoms in a single polypeptide chain.
Quaternary (4°) structure: The spatial arrangement of multiple polypeptide chains (subunits) in a protein complex.

Ramachandran Plots

Sterically Allowed Conformations

A Ramachandran plot maps the allowed values of φ and ψ angles for amino acid residues in a polypeptide.
White areas indicate sterically allowed conformations; shaded areas are forbidden due to atomic clashes.
Different secondary structures occupy characteristic regions on the plot.

Secondary Structure	φ (degrees)	ψ (degrees)
α-helix	~ -70 to -60	~ -50 to -40
β-sheet	~ -150 to -100	~ +120 to +160
Polyproline helix	~ -80 to -60	~ +130 to +140

Fibrous Proteins: Structural Materials of Cells and Tissues

Characteristics and Examples

Fibrous proteins are elongated, with well-defined secondary structures, providing structural support.
Examples include:
- Keratin: Found in hair, nails, feathers, and intermediate filaments.
- Fibroin: Main protein in silk, composed of β-sheets.
- Collagen: Most abundant connective tissue protein, forms triple helices and is a major component of bone matrix.

Keratin

Forms coiled-coil structures with hydrophobic interfaces.
Stabilized by repeating hydrophobic residues and disulfide bonds.

Fibroin

Composed of extensive β-sheets, providing strength and flexibility.
Interspersed with compact folded regions for elasticity.

Collagen

Triple-stranded left-handed helix, rich in proline and hydroxyproline.
Contains a repeating Gly-X-Y motif (X = Pro, Y = Pro or hydroxyproline).
Requires vitamin C for proper hydroxylation; deficiency leads to scurvy.

Globular Proteins: Tertiary Structure and Functional Diversity

General Features

Globular proteins are compact, with a hydrophobic core and hydrophilic surface.
Composed of varying amounts of α-helix, β-sheet, and loop regions.
Often organized into domains (~200 amino acids) that can fold and function independently.

Protein Domains

Domains are structural and functional units within proteins.
Each domain may have a specific function (e.g., DNA binding, enzymatic activity).

Common Features of Folded Globular Proteins

Nonpolar (hydrophobic) residues are buried in the interior; polar (hydrophilic) residues are exposed to solvent.
β-sheets are often twisted or form barrel structures.
Turns and loops allow the polypeptide chain to change direction.

Factors Determining Secondary and Tertiary Structure

Protein Folding Information

The amino acid sequence contains all the information required for folding into the native structure (Anfinsen's experiment).
Denaturation and refolding experiments demonstrate the reversibility of protein folding under suitable conditions.

Thermodynamics of Folding

Protein folding is governed by the Gibbs free energy equation:

Favorable enthalpic interactions: hydrogen bonds, ionic interactions, van der Waals forces.
Unfavorable entropy loss: folding reduces conformational entropy.
Hydrophobic effect: burying hydrophobic side chains increases solvent entropy, driving folding.

Stabilizing Interactions

Hydrogen bonds stabilize secondary and tertiary structures.
Disulfide bonds (covalent S–S bonds) greatly increase stability by reducing the number of possible unfolded conformations.
Binding of ions or prosthetic groups can further stabilize protein structure.

Dynamics and Kinetics of Protein Folding

Folding Pathways and Energy Landscapes

Protein folding follows a pathway from unfolded to native state, often via intermediates.
The energy landscape is often depicted as a funnel, with the native state at the bottom.
Some proteins may become trapped in misfolded states or aggregates.

Role of Chaperones

Chaperones are proteins that assist in the proper folding of other proteins, especially under stress conditions.
Chaperonins (e.g., GroEL/GroES in Escherichia coli) provide an isolated environment for folding.
ATP binding and hydrolysis drive conformational changes in chaperonins, promoting correct folding.

Protein Misfolding and Disease

Misfolded proteins can aggregate into amyloid fibrils, associated with diseases such as Alzheimer's, prion diseases, and type II diabetes.
Some misfolded proteins act as infectious agents by inducing misfolding in normal proteins (prions).

Quaternary Structure of Proteins

Organization and Symmetry

Quaternary structure refers to the arrangement of multiple polypeptide subunits in a protein complex.
Symmetrical arrangements (e.g., helical, point-group symmetries) are common in multisubunit proteins.
Interactions between different proteins (heterotypic interactions) are mediated by complementary surfaces.

Tools for Studying Protein Structure

Determining Molecular Mass and Subunit Composition

Size exclusion chromatography: separates proteins based on size.
Analytical ultracentrifugation: measures sedimentation to estimate mass.
Mass spectrometry: determines mass-to-charge ratios of protein components.
SDS-PAGE: separates protein subunits by size under denaturing conditions; reducing agents break disulfide bonds.

Technique	Main Purpose
Size exclusion chromatography	Estimate native molecular mass
SDS-PAGE	Determine number and mass of subunits
Mass spectrometry	Identify protein components

Summary

Protein structure is organized hierarchically from primary to quaternary levels.
Secondary structures (α-helix, β-sheet) are stabilized by hydrogen bonds and constrained by backbone geometry.
Folding is driven by thermodynamic principles and assisted by chaperones.
Misfolding can lead to disease, highlighting the importance of correct protein structure.
Biochemical techniques allow detailed analysis of protein structure and composition.